Protein glycosylation in Candida

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Héctor M Mora-Montes, Patricia Ponce-Noyola, Julio C Villagómez-Castro, Neil AR Gow, Arturo Flores-Carreón & Everardo López-Romero† Author for correspondence: Departamento de Biología, División de Ciencias Exactas y Naturales, Universidad de Guanajuato, Guanajuato, Gto. 36050, México n Tel.: +52 473 732 0006 ext. 8156 n Fax: +52 473 732 0006 n [email protected] †

Candidiasis is a significant cause of invasive human mycosis with associated mortality rates that are equivalent to, or worse than, those cited for most cases of bacterial septicemia. As a result, considerable efforts are being made to understand how the fungus invades host cells and to identify new targets for fungal chemotherapy. This has led to an increasing interest in Candida glycobiology, with an emphasis on the identification of enzymes essential for glycoprotein and adhesion metabolism, and the role of N ‑ and O ‑linked glycans in host recognition and virulence. Here, we refer to studies dealing with the identification and characterization of enzymes such as dolichol phosphate mannose synthase, dolichol phosphate glucose synthase and processing glycosidases and synthesis, structure and recognition of mannans and discuss recent findings in the context of Candida albicans pathogenesis. Candida albicans as a human pathogen Cell-wall components & their role in host-cell recognition

Candida albicans, a member of the microflora of mucosal surfaces and the digestive tract, is the most common cause of opportunistic fungal infections in immunocompromised patients. People who are predisposed to systemic candidiasis include premature infants, organ or bone marrow transplant recipients taking immunosuppressive drugs and patients undergoing invasive surgical procedures [1,2] . The mortality rate for systemic candidiasis has been reported as being between 14 and 90% in different patient groups, but is typically around 30–40% [3] . The incidence of fungal sepsis increased threefold between 1979 and 2000, with Candida spp. accounting for 70–90% of all invasive mycoses [4] . Immunocompromised patients treated with fungistatic drugs are often unable to completely clear the pathogen, which results in persistent cycles of fungal infection. Furthermore, some of the most effective antifungals have significant toxic side-effects. An understanding of the nature of the interaction between Candida spp. and the host may ultimately lead to the development of novel strategies for the control of candidiasis that combine appropriate chemo- and immunotherapies. The fungal factors underlying the pathogenic potential of this organism have been extensively investigated and these studies suggest that the pathogenesis of C. albicans is attributed to a set of virulence factors whose specific relevance depends on the precise setting and etiology of infections and the stage of progression of disease [5] .

Candida albicans cell-wall components play a major role in the interaction with the host cell at most stages of infection. The cell wall is the structure that first comes into contact with host cells of epithelia and of the immune system. It carries important antigenic determinants of the fungus, is responsible for the adherence of the pathogen to host surfaces and other micro organisms, and is vitally important for immune recognition and the activation of both the innate and adaptive immune responses [6] . Cell-surface adhesion proteins, generically known as adhesins, exhibit characteristics similar to those of lectins and integrins. They participate in mating, morphogenesis, biofilm formation and interactions with the mammalian hosts. Adhesins and other cell-surface components can be targeted by opsonizing antibodies, immune receptors and some antifungal drugs in order to interfere with adhesin function or with their anchorage in the cell wall. This interference can disrupt social networks among the fungi, preventing biofilm formation, and reduce the colonization of catheters and other foci of infection [7] . The Als proteins in C. albicans and EPA galectins in Candida glabrata represent two major families of adhesins whose biological roles have been extensively analysed [7] . Als proteins mediate adhesion to epithelia, yeast aggregation and colony and biofilm formation. Moreover, Als proteins, which probably evolved to facilitate mating, have been exploited in mediating interactions with host cells in both commensal and pathogenic situations [7] . In C. albicans, the Als family is an eight-membered gene family and each locus is heterozygous, containing trinucleotide repeat expansions that generate

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Future Microbiol. (2009) 4(9), 1167–1183

Review

Future Microbiology

Protein glycosylation in Candida

Keywords adhesin n Candida albicans cell wall n N‑glycan n protein glycosylation n virulence n n

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ISSN 1746-0913

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further phenotypic diversity [8] . Because of the redundancy and differential expression of different Als proteins in C. albicans, functions cannot always be assigned to single members of the family. However, Als1, which binds to endo thelia and epithelia, is the most widely expressed member of the family and this contributes to adhesion and colonization in an oropharyngeal candidiasis model [9] . Als3 is important for biofilm formation and induction of damage in an underlying oral epithelium [10] . For the Als proteins, the N‑terminal globular domain is necessary and sufficient for binding to cell surfaces. Als1 or Als5 confer C. albicans with the ability to adhere to endothelia and epithelia, as well as to biochemically defined substrates [11–15] . Candida albicans Hwp1, a glutamine-rich, glycosylphosphatidylinositol (GPI) wall-anchored adhesin expressed on hyphal cell walls, is a substrate for epithelial cell transglutaminases that can covalently crosslink Hwp1 to epithelial proteins. The resulting association generates shearresistant, closely adherent interactions with host surfaces. Such interactions could underlie the clinical observation that oropharyngeal colonies of C. albicans resist removal by scraping [16] and may account for the persistence and long-term carriage of C. albicans on mucosal surfaces. The functions of these proteins have been investigated using biochemical and genetic methods. Mutants with specific alterations in the cell surface have proved to be valuable tools in determining which surface components are recognized by immune cells. Components within the yeast cell wall that represent pathogen-associated molecular patterns have been defined through the use of mutants with defects in cell-wall glycosylation and in the biosynthetic pathways leading to the assembly of glucans and chitin, which are present in the deeper layers of the cell wall. The glycosylation mutants that have been used have alterations in saccharidic moieties of mannan, mannoproteins (MPs) and glycolipids – all of which are expressed at the cell-wall surface [17] . Cell-wall MPs have been widely investigated as major players in host–pathogen relationships [18,19] . Surface-located and secreted MPs are involved in cell–cell recognition and responses to stress factors and subsets of proteins with specific virulence-related functions. Some MPs are important for the adhesion of the fungus to the host-cell surface – the essential first step in pathogenicity and disease transmission – and they may also be involved directly in the activation of the adaptive humoral and cell-mediated 1168

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immune responses of the host [5] . The host immune system includes receptor recognition of molecules that are present in both the superficial and deeper layers of the fungal cell wall. For example, some pattern-recognition receptors recognize the outer MP layer, while dectin‑1 recognizes cell-wall b1,3‑glucan, a potent pro inflammatory molecule that is found in the inner layers of the C. albicans cell wall [20,21] . Functions of fungal-wall glycoproteins include maintenance of cell-wall integrity and cell-wall remodeling, homotypic and heterotypic adhesion, biofilm formation, acquisition of iron and sterols, protein degradation and coping with oxidative stress [22,23] . Transcriptome studies indicate that the expression levels of genes encoding MPs is highly variable but often tightly controlled; for example, they include genes that are highly regulated at the transcriptional level during the yeastto-hypha transition [24] . However, owing to their complex and variable glycosylation, fungal-wall glycoproteins are difficult to analyze using traditional proteomic approaches. Recent advances in mass spectrometry-based proteomics have enabled rapid and sensitive identification and quantitation of fungal-wall glycoproteins [23] . These approaches will be particularly useful for studying the dynamics of the subproteome of fungal-wall glycoproteins, and for the development of novel vaccines and diagnostic tools [6] . Because glycoproteins are therefore of indisputable importance in fungal pathogenesis, much of the effort of our groups and others has focused on the study of glycoprotein biosynthesis, with an emphasis on specific glycosyltransferases and glycosidases, and their role in C. albicans virulence. Many basic aspects of cell-wall glycosylation have been illuminated in Saccharomyces cerevisiae, in which the events of assembly and secretion of glycoproteins have been extensively studied [25,26] . Information obtained from S. cerevisiae and pathogenic fungal species will hopefully lead to a better understanding of infections and help in the long-term goal of designing new diagnostic and therapeutic approaches against fungal disease. Mannosyl & glucosyl transferases Dolichol phosphate mannose synthase

Several protein glycosylation pathways take place in the lumen of the endoplasmic reticulum (ER), where mannose and glucose residues are sequentially added to acceptor molecules from dolichol phosphate (DP) sugars by specific glycosyltransferases. Mannosyl transfer from DP mannose (DPM) in the ER lumen is involved future science group


in protein N‑, O‑ and C‑glycosylation as well as in the synthesis of GPI anchors. N‑linked glycosylation involves attachment of high molecular weight, highly branched glycans to asparagine residues via amide, while O‑linked glycosylation involves the attachment of shorter linear glycans via ether linkages to serine or threonine residues of polypeptides. C‑mannosylation is a novel type of protein glycosylation that involves the covalent attachment of an a‑mannopyranosyl residue to the indole C2 carbon atom of tryptophan via a C–C link [27,28] . Mannosylation has been shown to be essential for many organisms. For example, deficient mannosylation can result in severe forms of congenital disorders of glycosylation (CDG) [29–31] , frequently with a lethal outcome. Also, failure to synthesize GPI causes death in mice [32] , and is an essential process in S. cer‑ evisiae [33,34] and Trypanosoma brucei [35] . DPM is widely used for glycosylation and is the only mannose donor for mannosylation in the lumen of ER, as this organelle lacks a transporter for GDP–Man – the mannosyl donor used by sugar nucleotide-specific glycosyltransferases such as those that operate in the cytosolic side of ER [36] . By contrast, the Golgi apparatus, where elongation of O‑linked sugar chains by GDP–Mandependent mannosyl transferases (MTs) and processing of complex N‑linked oligosaccharide structures take place, does contain a GDP–Man transporter [37,38] , which couples the entry of cytosolic sugar nucleotide into the Golgi lumen to the exit of equimolar amounts of GMP, which is produced from GDP by the action of a luminal GDPase encoded by the GDA1 gene in C. albi‑ cans and S. cerevisiae [39,40] . The Golgi nucleotidase influences morphogenesis, glycosylation and cell-wall properties in C. albicans [40] . Synthesis of DPM is catalyzed by DPM synthase (DPMS), which transfers mannose from GDP–Man to the ER membrane-anchored polyisoprenoid DP. DPMS is a member of glycosyltransferase family 2 and a specific marker for the ER. In humans, lack of DPMS causes CDG‑1e [41,42] , whereas defects in enzymes related to the synthesis or use of DPM result in a number of other related CDGs [43] . Most of what we presently know about fungal DPMS comes from studies carried out in S. cerevisiae where the enzyme gene, DPM1, was shown to be essential for viability [44] . Later, homologs to yeast DPM1 were identified in T. brucei [45] , Ustilago maydis [46] , Schizosaccharomyces pombe [47] , Caenorhabditis briggsiae [47] , humans and mice [47,48] . The Dpm1 proteins fall into two classes. The first class carries future science group

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a hydrophobic sequence at the C‑terminus and includes S. cerevisiae, U. maydis, T. brucei and Leishmania mexicana [49] . In the second class, which includes enzymes from S. pombe, C. brigg‑ siae, Trichoderma reesei and mammalian cells [50] , the Dpm1 proteins lack a hydrophobic domain and thus appear to be soluble. Recently, structural studies led to a 3D model that helps to explain the catalytic mechanism of yeast DPMS [51] . In addition, elegant studies by Maeda’s group demonstrated that mammalian DPMS is a complex consisting of three proteins, Dpm1, Dpm2 and Dpm3, each with an essential and specific function in enzyme operation. Dpm1 is the catalytic component and is bound, via its C‑terminus, to the membrane-anchored Dpm3, which tethers it to the ER membrane and allows a stable enzyme activity, while Dpm2 interacts with and stabilizes Dpm3 [48,52,53] . Because DPMS is a potential antifungal target, we have studied this enzyme in C. albicans, mostly with the objective of defining the functional interaction of DMPS with other MTs, the nature of products formed by the sugar transfer reactions and the specific requirements for these reactions to occur both in C. albicans membrane and soluble fractions. An assay was developed using mixed membrane fractions (MMFs) obtained from C. albi‑ cans that used GDP–14C–Man to form DPM. The sugar from this intermediate was transferred efficiently onto MP by protein MTs (PMTs) in functionally coupled reactions. This reaction was stimulated four- to fivefold by exogenous DP. Low amounts of lipid‑linked oligosaccharides were also detected. Nearly all sugar in the MP was O‑linked and mannose was the sole sugar released after b‑elimination. Specific inhibition of DPMS with amphomycin showed that all sugar was transferred to protein via DPM. Interestingly, substitution of radiolabeled GDP– Man by exogenous 14C–DPM failed to mannosylate proteins to a significant extent, implying that a close topological and functional relationship between dolichol-dependent MTs exists in the ER membrane that allows an efficient use of endogenously generated DPM as the mannosyl donor by PMT [54] . It is well documented that PMT catalyzes the first O‑mannosylation step of secretory proteins in the ER by adding a mannose residue from DP to the hydroxy group of Ser and Thr hydroxy amino acids. Glycoproteins are then transported to the Golgi apparatus, where O‑linked sugar chains are elongated by specific, luminal sugar nucleotide-dependent glycosyltransferases [18,23,37,38,55,56] . C. albicans www.futuremedicine.com

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O‑glycans consist of up to five a1,2‑linked mannose chains [40] . The genome of this pathogen contains five PMT genes, of which PMT1 and PMT2 have been characterized [57,58] . PMT proteins share high homologies among themselves and with S. cerevisiae orthologs, and it has been shown that Pmt2, as well as the combined action of Pmt1 and Pmt4, are required for growth of C. albicans in all conditions [18] . For an excellent review of protein O‑mannosylation, see [23] . Based on this knowledge, failure to detect mannobiose and/or mannooligosaccharides following incubation of C. albicans membranes with radiolabeled GDP–Man reflects the inability of these fractions, as well as detergent-extracted enzyme preparations (see later), to elongate monomannosylated MP. This is presumably owing to the requirement of particular conditions in the ER and Golgi that are not met in these in vitro reactions, or to the inactivation of Golgi MTs during preparation of enzyme fractions. The simultaneous, tandem operation of DPMS and PMT in the same enzyme preparation complicates the study of individual MT reactions. To overcome this, several attempts have been made to selectively extract the enzymes from MMF and then purify them. At first, purification of DPMS seemed less problematic than that of PMT, as activity of the latter depended on that of the former. To study PMT, it was necessary to obtain a soluble preparation with the ability to transfer measurable amounts of radioactivity from exo genous 14C–DPM to proteins. To this end, several detergents and incubation conditions were used for selective enzyme extraction. Best results were invariably obtained using the nonionic surfactant Nonidet®‑P40 (NP‑40). As summarized in Table 1 [59–62] , the concentration of NP‑40 used to extract the membranes had a strong effect on activity and catalytic properties of solubilized MTs. After several attempts to separate DPMS and PMT, it was shown that treatment of MMF with NP‑40 at a detergent/protein (D/P) ratio of 0.30 released a solubilized fraction (SF) enriched in DPMS and virtually free of PMT. The stability of this fraction allowed the partial purification of DPMS by a single step of preparative nondenaturing electrophoresis and enzyme characterization, with emphasis on regulation by phosphorylation [62] . Accordingly, accumulated evidence indicates that DPMS from organisms as divergent as fungi [63] , protozoans [64] and mammals [65,66] can be activated in vitro by phosphorylation to levels that range from 40–80% in rat microsomes [65,66] to 40–380% in Entamoeba histolytica [64] . These levels of activation correlate with an increase in 1170


maximum velocity (Vmax) with no effect on the affinity for GDP–Man. In agreement with these findings, the C. albicans DPMS was activated up to 60% by cAMP-mediated protein phosphorylation and this activation correlated with a 50% increase in Vmax with no change in the apparent Michaelis–Menten constant (K m) for GDP– Man. Phosphorylation with [g‑32P]ATP labeled several polypeptides, one of which exhibited the enzyme molecular mass (30 kDa) and was recognized by a specific anti-DPMS monoclonal antibody [62] . The significance of this regulation by phosphorylation–dephosphorylation remains to be established. Partially purified PMT exhibits different phospholipid requirements with respect to DPMS, suggesting that although they are tightly coupled in the ER, the hydrophobic environment of each enzyme is provided by different membrane components. More importantly, coupled transfer activity between the MTs could be reconstituted in vitro by mixing the partially purified DPMS and PMT, indicating that this process can occur in the absence of cell membranes [67] . DP glucose synthase

Dolichol phosphate glucose (DPG) synthase (DPGS) catalyzes the transfer of glucose from UDP–Glc into DP, forming DPG, a glucosyl donor for the assembly of Dol–P–P–GlcNAc2 – Man9 Glc 3, the oligosaccharide portion of which is transferred to nascent proteins in the ER lumen by the oligosaccharyl transferase (OST) complex [25,68] . Yeast mutants unable to form DPG transfer underglycosylated oligo saccharides in vivo [69–71] . Mutant complementation studies have led to the isolation of the ALG5 locus encoding for the transferase [70] . It is claimed that the glucotriose unit in the Dol–P–P–GlcNAc2 –Man9Glc3 intermediate is important not only for glycosylation efficiency but also for the recognition of the glycosylatable Asn–Xaa–Ser/Thr sequons [72] . Photoaffinity labeling studies indicated that the catalytic subunit of the enzyme is associated with a transmembrane 35‑kDa polypeptide in yeast [73] and rat-liver microsomes [74] , and with a 39-kDa protein in mung-bean cells [75] . Similar approaches have yet to be carried out in C. albicans. Membranes from C. albicans also catalyzed the transfer of glucose from UDP–14C–Glc, mainly into DPG (80%) and minor proportions of glycoprotein and lipid‑linked oligosaccharides, with each acceptor representing approximately 10% of total transferred radioactivity. Conditions that stimulated or inhibited DPG synthesis did future science group


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Table 1. Summary of mixed membrane fraction extraction experiments and properties of solubilized mannosyl transferases. D/P Mannosyl transfer properties of solubilized fraction ratio*

Comments

0.025

A coupled complex consisting of DPMS, PMT and DP was solubilized Some general properties of mannosyl transfer reaction were investigated

0.063

0.30 1.4 0.09

Exogenous DP-independent sequential transfer of mannose from GDP–Man into DP and MP Catalytic activity of PMT seemed higher than that of DPMS Efficient use of exogenous 14C–DPM as sugar donor MP contained a single mannose residue Fully exogenous DP-dependent transfer of mannose from GDP–Man into DP (84%) and MP (16%) Mannosyl transfer activity was instable Similar to that released at 0.063 but DPMS was remarkably more stable Efficient transfer of mannose from exogenous 14C–DPM into endogenous protein acceptors SF catalyzed glucolipid synthesis in the absence of exogenous DP

DPMS and PMT were solubilized and uncoupled from DP Allowed the purification and partial characterization of DPMS Allowed the partial purification and characterization of PMT Allowed the partial characterization of DPGS and comparison of properties with the MMF-bound enzyme

Ref. [59]

[60,61]

[62] [67] [60]

*Nonidet ®-P40 (NP‑40) was used in all experiments at the indicated D/P ratios. A D/P ratio of 0.025 corresponds to 0.016–0.019% (w/v) or 0.26–0.30 mM. The critical micellar concentration of NP‑40 is 0.25 mM. Since NP‑40 inhibited mannosyl transfer activity, it was removed by treating solubilized fractions with hydrophobic adsorbent beads prior to assay of enzyme activity. D/P: Detergent/protein; DP: Dolichol phosphate; DPM: DP mannose; DPMS: DPM synthase; MMF: Mixed membrane fraction; MP: Mannoprotein; PMT: Protein mannosyl transferase; SF: Solubilized fraction.

not affect glycoprotein labeling, suggesting that this resulted from the direct transfer of glucose from UDP–Glc by glucosyl transferases released from Golgi vesicles [76] . Recovery of most of the glucose in DPG was expected because it is known that DPG is used by ER-specific glucosyltransferases for the addition of three glucose residues to dolichol–PP-anchored M9 prior to transfer of the oligosaccharide to nascent proteins by the OST complex. Shortly after this transferase reaction, glucose is removed from the developing triantennary complex by specific glucosidases I and II, as will be discussed later. Gold and Green observed that DPG synthesis in mammalian cells was inhibited by millimolar concentrations of UMP, UDP, GTP and UTP [77] . Similarly, we found that ATP, GTP, UTP, ADP and UDP strongly inhibited C. albicans DPGS in the range of 1.25 to 2.5 mM. GMP had no effect, while UMP was a competitive inhibitor with an affinity for DPGS closely comparable to that of GDP–Glc. Upon incubation with radiolabeled DPG, UMP (but not UDP) was able to reverse the DPGS reaction up to 57% at 0.62 mM [76] . These results are consistent with previous observations for DPGS from mammalian [78] , plant [79] and protozoan [80] cells. The role of nucleotides in the regulation of DPGS in vivo remains to be elucidated. As shown in Table 1, incubation of MMF with NP‑40 at a D/P ratio as low as 0.09 released an active SF that catalyzed glucolipid synthesis in the absence of exogenous DP. This fraction allowed the determination and comparison of catalytic properties of solubilized future science group

glucosyl transferase with those of the particulate DPGS [60] and also with the enzyme from other organisms [75,77] . Glucose transfer by the solubilized DPGS was threefold more sensitive to inhibition by amphomycin, a specific inhibitor of DP‑dependent glycosyl transferases [81] , than the particulate enzyme. Re-examination of the effect of nucleotides confirmed the competitive inhibition of DPGS by UMP and also an essentially similar dependence of cations for activity, namely, the requirement of Mg2+ and the slightly inhibitory effect of Ca 2+ and Mn2+ [60] . This was the first report on the identification and characterization of DPGS in a pathogenic dimorphic fungus. Clearly, further studies will be necessary to refine our knowledge of the structure, mechanism of operation and regulation of DPMS, DPGS and other equally important glycosyl transferases in C. albicans and other pathogenic organisms. N‑glycan processing a‑glycosidases

As mentioned above, the dolichol pyrophosphateanchored oligosaccharide Glc3Man9GlcNAc2 (Glc3M9) is transferred to an Asn residue on nascent polypeptides by the ER OST complex. In yeast, this complex consists of at least eight subunits: Ost1, Ost2, Ost3 (or Ost6), Ost4, Ost5, Wbp1, Swp1 and Stt3. STT3, OST1, OST2, SWP1 and WBP1 are essential for growth, suggesting a role in the catalytic activity of the complex [82] . Much less is known about this enzyme complex in C. albicans. However, the availability of its genome sequence [83] will www.futuremedicine.com

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facilitate in silico methods to identify putative genes encoding OST subunits. After transfer, Glc3M9 is trimmed by glucosidases I and II and mannosidase I [25,84–86] . Glucosidase I, a type II membrane protein with a luminal catalytic domain [87] , removes the outermost a1,2‑linked glucose to yield the Glc2Man9GlcNAc2 (Glc2M9) oligosaccharide, which is further trimmed by glucosidase II, which removes the middle and innermost a1,3‑linked glucose residues, yielding Glc1Man9GlcNAc2 (Glc1M9) and Man9GlcNAc2 (M9) forms, respectively (Figure 1) . These early steps of N‑linked oligosaccharide processing are similar in yeast, plant and mammalian cells [88] . Finally, ER mannosidase I selectively removes a specific mannose residue, yielding the Man8GlcNAc2 (M8) oligosaccharide before glycoproteins either exit the ER or are targeted for degradation if they fail to fold properly (Figure 1) . In yeast, this specific mannose removal induces a conformational change of the oligosaccharide that facilitates outer-chain synthesis [89] . In mammalian cells, several mannose residues are removed by different mannosidases. Hydrolysis of a1,2‑linked mannose residues gives rise to hybrid and high-mannose N‑glycans, whereas removal of a1,3‑ and a1,6‑linked mannoses leads to formation of complex N‑glycans [68,90] .

a‑glucosidases I & II

a‑glucosidase I from S. cerevisiae has been obtained as both membrane-bound and soluble forms. The latter has been purified to homogeneity and characterized [91] . The yeast CWH41 gene encodes the processing a‑glucosidase I (Cwh41) [92] , which is a member of family 63 of glycosyl hydrolases [201] . This enzyme is a type II membrane protein with a proposed domain orientation comparable to its mammalian counterpart [87,93,94] . Sequence comparison between enzyme orthologs (i.e., human, Caenorhabditis elegans and S. cerevisiae) shows higher identity (34–49%) at the end of the C‑terminus domain where the putative catalytic pocket is located [92] . Indeed, proteolysis of membrane-bound mammalian and soluble yeast a‑glucosidase I released catalytically active polypeptide(s) from the luminal domain [87,95,96] . a‑glucosidase I catalysis, like that of other glycosidases, is controlled by specific carboxy amino acid residues [97] . Based on chemical modification, other residues, such as Arg, Cys and Trp, were reported to be likely participants in the binding site of mammalian a‑glucosidase I [98,99] . Dhanawansa et al. showed that yeast a‑glucosidase I is sensitive to modification of His but not of Cys, distinguishing it from

1A 1B 1C

GI

Mns1

GII

ER lumen

Cytosol

Ribosome

Man

Polypeptide chain

Glc

Folded protein GlcNAc

α1,3-

α1,6-

Linkage type

α1,2-

Future Microbiol. © Future Science Group 2009

Figure 1. Processing of Glc3Man9GlcNAc2 oligosaccharide by endoplasmic reticulum glycosidases. ER: Endoplasmic reticulum; GI: Glucosidase I; GII: Glucosidase II; Mns1: Mannosidase 1.

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the mammalian enzyme [97] . Site-specific chemical modification of the soluble a‑glucosidase I from yeast using diethylpyrocarbonate and tetranitromethane revealed that His and Tyr are involved in enzyme activity [100,101] . A fragment of C. albicans CWH41 was amplified and then expressed in E. coli as a soluble 50‑kDa polypeptide with a‑glucosidase activity on 4‑methylumbelliferyl-a‑d‑glucopyranoside, a fluorogenic substrate not usually cleaved by a‑glucosidase I [95] . Yet, this substrate has been useful for the characterization of this enzyme in C. albicans [102] . Glucosidase II has been extensively characterized from mammalian tissues as a soluble enzyme consisting of two tightly associated a‑ and b‑glycoprotein chains [103,104] . The a-subunit (GIIa) is a 107-kDa protein that shares sequence similarity with other glucosidase enzymes and exhibits glycosyl hydrolase activity in vitro that is independent of the b‑subunit (GIIb) [104] . The function of GIIb is not well defined but it has been suggested that it may be required to retain the heterodimer in the ER via its C‑terminal HDEL motif [105,106] . In addition, GIIb may interact with N‑glycans via a carbohydrate recognition domain to stimulate the trimming of both the middle and innermost glucose residues by GIIa [107] . Glucosidase II has been characterized in S. cerevisiae, where the GLS2 gene has been shown to encode a GIIa homolog (Gls2) and gls2 null mutants are unable to process the Glc2M9 oligosaccharide [103,108] . More recently, Wilkinson et al. reported that an uncharacterized open reading frame, YDR221w, encodes the yeast homolog of GIIb [109]. This open reading frame, named as GTB1, expresses a soluble 96–102‑kDa glycoprotein that coimmunoprecipitates with Gls2. The same authors also observed that the stability and localization of Gls2 was not altered in a gtb1 null mutant, suggesting that yeast Gtb1 is not required for retention of Gls2 in the ER. Furthermore, the trimming of Glc2M9 to Glc1M9 was unaffected in this mutant, demonstrating that Gls2 is sufficient for the reaction. However, the gtb1 cells accumulated monoglucosylated forms of N‑linked glycoproteins, indicating that Gtb1 is specifically required for the final glucose-trimming event during normal glycoprotein processing. A soluble a‑glucosidase from C. albicans yeast cells was purified by a three-step procedure consisting of size-exclusion, ion-exchange and adsorption chromatographies. The purified preparation revealed the presence of two major polypeptides of 36 and 47 kDa, the latter being future science group

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responsible for activity. The enzyme’s ability to convert the substrate GlcMan9GlcNAc2 into the M9 product, as well as other biochemical properties, were consistent with an a‑glucosidase II being involved in N-glycan processing [110] . Because the molecular mass of the protein is lower than that predicted for CaRot2 (the homolog to S. cerevisiae Gls2), it is proposed that the a‑glucosidase II polypeptide is proteolytically processed during cell disruption, generating a soluble catalytic domain. a‑mannosidases

Based on amino acid sequence analysis and some biochemical properties, a‑mannosidases are grouped into glycosyl hydrolase families 38 (a1,2‑, a1,3‑ and a1,6‑mannosidases) and 47 (which includes a1,2‑mannosidases only) [85,90,111] . Depending on substrate specificity, two types of a1,2‑mannosidases can be recognized within family 47: the ER enzymes that eliminate one mannose residue from M9 to form M8 isomer B (M8B) [112,113] , and the Golgi-resident a‑mannosidases IA, IB and IC, which trim the four a1,2‑linked mannoses from M9 to produce Man5GlcNAc2 [114,115] . In addition, evidence supports the involvement of a1,2‑mannosidases in the demannosylation of intermediate oligo saccharides during the process of ER-associated glycoprotein degradation [55,116,117] . Studies of a‑mannosidase activity in C. albi‑ cans American Type Culture Collection (ATCC) 26555 showed that approximately 80% of the total cellular activity was present as a soluble form (Figure 2) [118] . Purification of the enzyme from cell extracts obtained in the presence of the protease inhibitor phenylmethanesulfonyl fluoride led to the separation of low amounts of two enzyme fractions, named E‑I and E‑II, whose relative proportion varied in the different experiments. E‑I and E‑II preferentially released mannose from a1,6‑linked mannan and a1,3‑linked mannobioside, respectively [119] , and converted the processing-inert Man10GlcNAc oligosaccharide into a mannoseaccepting substrate [120] . Further experiments in the absence of phenylmethylsulfonylfluoride allowed purification of E‑II, which, under these conditions, represented up to 70% of total activity (Figure 2) . Analytical electrophoresis of purified E‑II revealed a single polypeptide of 43 kDa that was responsible for a‑mannosidase activity as judged from zymogram analysis [121] . This molecular mass is close to the 45 kDa reported for a soluble a‑mannosidase purified from S. cerevisiae [122] . Purified E‑II acted on www.futuremedicine.com

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ATCC 26555

CAI4

Partial aneuploidy in chromosome IV

Strain of molecular reference (ura- mutant)

- PMSF

+ PMSF

Soluble

80%

47%

Soluble

47%

E-I (52 kDa)

30%*

100%

E-I*

100%

E-II (43 kDa)

70%*

MMF-bound (65 kDa)

20%

0 53%

- PMSF

E-II MMF-bound

0 53%

Figure 2. Distribution of a-mannosidase activity in Candida albicans. *With respect to total soluble activity. ATCC: American Type Culture Collection; MMF: Mixed membrane fraction; PMSF: Phenylmethanesulfonyl fluoride.

M9 as a typical ER-resident a1,2‑mannosidase [86] , producing M8B and mannose as the sole products of hydrolysis after 12 h of incubation and was also active on M8B, releasing M7 and probably M6. Shorter oligosaccharides, such as Man6GlcNAc2 –Asn, Man5GlcNAc2 –Asn and a1,3‑ and a1,6‑linked mannobiosides, were not cleaved at all. These properties are consistent with an a1,2‑mannosidase belonging to glycosyl hydrolase family 47 [121] . Purification of soluble a‑mannosidase from C. albicans CAI4, a strain with a different genetic background from ATCC 26555 [123,124] , revealed the presence of only E‑I, indicating the inability of this strain to produce E‑II. The identification of a‑mannosidases E‑I and E‑II made C. albicans an attractive model to study fundamental steps of protein N‑glycosylation, as it has been described that lower eukaryotes contain only one membranebound N-glycan processing a‑mannosidase [85,86] . To investigate whether soluble a‑mannosidase could be released by proteolysis of the enzyme present in the MMF, cell disruption was carried out in the presence of the protease inhibitor pepstatin A. This condition produced membrane fractions showing increased amounts of a‑mannosidase at the expense of a proportional decrease of soluble activity, which was solely conformed by enzyme E‑I. These results suggested that E‑II may be generated by a proteolytic event occurring during cell disruption. Differential density gradient centrifugation of 1174


cell homogenates revealed that E‑I was localized within the cytosolic fraction and Golgi-derived vesicles, and that a 65‑kDa membrane-bound a1,2‑mannosidase was present in ER- and Golgiderived vesicles [125] . It was also demonstrated that the membrane-bound a1,2‑mannosidase is converted into E‑I by the Kex2 protease, which recognizes an atypical cleavage site in the precursor sequence [125] . Limited proteolysis by Kex2 may represent a regulatory mechanism similar to that recently described for the human ER a1,2‑mannosidase [126] . Analysis of cytosolic free N‑oligosaccharides indicated that cytosolic a1,2‑mannosidase E‑I trims free M8B into M7B [125] . Moreover, antibodies raised against either the 43- or 52-kDa recombinant protein recognized all enzyme species, indicating that a‑mannosidases E‑I, E‑II and the membrane-bound form are immunorelated. This relationship was further supported by the observation that a C. albicans mns1D null mutant lacks soluble and membrane-bound a‑mannosidase activities [102] . To further investigate the ER a1,2‑mannosidase, the MNS1 gene from C. albicans was cloned in E. coli, expressed and the enzyme purified [127] . The biochemical properties of the recombinant enzyme were closely comparable to those of Mns1 isolated from C. albicans membranes [127] . In S. cerevisiae, CWH41, ROT2 and MNS1 genes encode for a‑glucosidase I, the catalytic subunit of a‑glucosidase II and a1,2‑mannosidase, respectively. When the homologous genes in C. albicans were disrupted, the resulting null mutants Cacwh41D, Carot2D and Camns1D tended to aggregate, displayed reduced growth rates, had a lower content of cell-wall phosphomannan and experienced other changes in cell-wall composition. These mutants under glycosylated b‑N‑acetylglucosaminidase, and had a constitutively activated PKC–Mkc1 cellwall integrity pathway [102] . Disruptants were also attenuated in virulence in the murine model for systemic candidiasis and stimulated an altered pro- and anti-inflammatory cytokine profile in human monocytes. These results strongly suggest that processing of C. albicans N‑glycans by ER glycosidases is required for cell-wall integrity and host–fungus interaction [102] . N‑ and O‑linked mannans & the C. albicans–host interaction

The assembly of N‑ and O‑linked mannans was first dissected and characterized in S. cerevisiae. However, this yeast is not an appropriate model for studies of the host–fungus interactions, since it is virtually nonpathogenic and its ancestor future science group


diverged from C. albicans over 200 million years ago [128] . Thus, the study of the mannosylation pathways in pathogens such as C. albicans is an attractive area to establish the specific importance of mannans for virulence and fungal immune recognition. The generation of C. albicans null mutants lacking key genes for mannan synthesis has demonstrated that both N‑ and O‑linked mannosylation are required for virulence. Loss of ER glycosidases (Cwh41, Rot2 and Mns1) [102] , protein mannosyltransferases (Pmt1, Pmt2, Pmt4, Pmt5 and Pmt6) [18,57,58,129] , Golgi mannosyltransferases (Och1, Mnn5, Mnt1 and Mnt2) [130–133] or depletion of Mn 2+ levels within the Golgi complex [134] leads to an attenuation of virulence in the murine model of systemic candidiasis. Many of these mutants show defects in growth rates in vitro [57,102,130,135] and some genes that encode proteins required for mannan synthesis are essential for C. albicans viability [18,136,137] . Since defects in the C. albicans growth rate are strongly correlated with attenuation of virulence [138] , it is conceivable that the growth defects alone account for the virulence defect in the mannosylation mutants. However, these mutants also show changes in cell-wall composition, have increased sensitivity to cell-wall-perturbing agents such as calcofluor white, Congo red, caffeine, tunycamycin, hygromycin B and vanadate and often exhibit constitutive activation of the cell-wall-integrity pathway and delayed filamentation [18,57,102,130–137] . Thus, severe defects in N‑linked mannan biosynthesis have a negative impact on overall cell fitness, which is highly likely to relate to the observed attenuation in virulence. However, not all mannosylation mutants have a strong in vitro phenotype. For example, the C. albicans mnn4D null mutant is unable to add phosphomannan to both N‑linked and O‑linked mannans, and the virulence, cell growth, morphogenesis and cell-wall assembly of these strains are not affected [139] . Most mutations in individual glycosyltransferase genes lead to weak phenotypes – perhaps owing to some redundancy of function within the families encoding many of the major glycosylation steps. Many of the proteins required for C. albicans adhesion are highly mannosylated [140–144] , and these post-translational modifications are required for protein folding, secretion and biofilm production [145] . Therefore, C. albi‑ cans cells that have disruptions in key steps in the mannosylation pathway may be affected in the folding, transport and activity of key virulence factors in the cell wall. future science group

Review

Most of the cell-wall MPs are modified with GPI anchors at the C‑terminus. This posttranslational modification is required for the attachment of most of the MPs to the cell wall and, thus, also has a vital role in cell viability and wall integrity [146,147] . For example, repression of SMP3 expression, which encodes an essential mannosyltransferase for GPI elaboration, leads to cell-wall defects and loss of cell viability [148] . C. albicans mutants with defects in the biosynthesis of chitin [149,150] , b1,3‑ and b1,6‑glucans [151–153] have shown that these cell-wall components are important for cellwall integrity, cell viability and for virulence. Therefore, cell-wall homeostasis is a complex property of the integrated assembly of many cell-wall polysaccharides and is essential for C. albicans viability and virulence. Immune recognition of C. albicans glycans

Mannans, chitin, b1,3‑ and b1,6‑glucans are the major components of the C. albicans cell wall. It has been demonstrated that b‑glucans and mannans are pathogen-associated molecular patterns recognized by pattern-recognition receptors expressed on the surface of immune cells [154–161] . However, the relative contribution of N‑linked mannans to C. albicans immune recognition has only recently been explored. Recognition of N‑linked mannans depends upon their structure and the type of immune cells interacting with the fungal cell. The mannose receptor (MR), a C‑type lectin expressed on the surface of dendritic cells and macrophages, recognizes branched oligos accharides containing a‑linked mannose residues, such as the C. albicans N‑linked mannans [162] . Recently, Netea et al. demonstrated that MR is the main receptor for N‑linked mannans during immune recognition of C. albicans by macrophages and monocytes [159] . Cytokine production by these immune cells was significantly reduced upon stimulation with C. albicans och1D null mutant. The cell wall of this mutant lacks N‑linked mannan outer chains [130] , and recognition of C. albicans wild-type, but not of och1D null mutant, was significantly blocked by anti-MR antibodies [159] . Similar observations have been obtained using C. albicans rot2D and cwh41D null mutants, which also harbor severe defects in N‑linked mannosylation [102] . However, mutants displaying an intermediate defect in the outer-chain elaboration, such as the mns1D null mutant, or lacking phosphomannan, www.futuremedicine.com

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stimulated normal cytokine responses, indicating that these N‑linked mannans were still recognized by immune cells [102,159] . Thus, it is likely that the branches of N‑linked mannan outer chain, composed of a1,2- and a1,3‑mannose residues, represent the epitopes recognized by MR. C. albicans recognition by dendritic cells requires a second receptor, the C‑type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing-nonintegrin (DC‑SIGN) [163–166] . As in the case of MR, DC‑SIGN only recognized the a1,2- and a1,3‑mannose units of the N‑linked mannan branches [166] . While MR and DC‑SIGN are involved in the recognition of a‑linked mannose, b1,2-mannosides, present in both acid-labile and -stable fractions of N‑linked mannans [167] , are recognized by the galectin‑3 receptor [168] . The b1,2‑mannose units are also associated with glycophospho lipids named phospholipomannans, and these carbohydrates are not present in S. cerevisiae, suggesting that these sugar residues may confer special attributes to C. albicans relevant to the interaction with the host immune system. Supporting this, it has been shown that b1,2‑mannose sensing through galectin‑3 and TLR2 helps macrophages to discriminate between C. albicans and S. cerevisiae [168] . However, the recognition of b1,2‑mannose residues seems to be nonessential for monocyte recognition, because C. albicans cells lacking b1,2‑mannose units induce similar cytokine production to cells with these sugar moieties presented at the surface [Mora-Montes & Gow, Unpublished Data] . Nevertheless, b1,2‑mannose residues are required for C. albicans adhesion to host tissues [169,170] and for generation of protective antibodies against systemic and vaginal candidiasis [171,172] . Recently, the C‑type lectin Mincle has been shown to be an important fungal pattern-recognition receptor on macrophages [173,174] . This lectin probably also binds to mannose-based polymers of the cell surface, although the precise nature of the ligand remains to be defined. Another C‑type lectin involved in the recognition of C. albicans N‑linked mannans is dectin‑2 [175–177] . This receptor is expressed on the surface of dendritic cells and macrophages, and shows preferential binding to C. albicans hyphae over yeast cells [175,176] . This suggests that N‑linked mannan structures on the hyphal surface may be different from those present on the yeast cell wall. Dectin‑2 lacks a cytoplasmic domain and must interact with FcRg to be able to induce cytokine production [176] . 1176


Recently, it has been proposed that in the mouse model for systemic candidiasis, the damage to kidney tissue is directly related to the intensity of the host innate immune response against C. albicans [178] . Virulent strains stimulated higher cytokine and chemokine levels than strains with attenuated virulence, which correlated with higher levels of immune cells infiltrate in kidney lesions and greater tissue damage caused by inflammation [178] . N‑ and O‑linked mannans, along with b1,3‑glucan, are the main C. albicans cell-wall components that stimulate proinflammatory cytokine production in monocytes [159,160] . Thus, it is possible to draw a direct relationship between mannan structure, activation of immune response and virulence. Mutants with severe defects in mannan assembly (e.g., mnt1D/mnt2D, och1D, pmr1D, rot2D and cwh41D), stimulate lower levels of proinflammatory cytokines and are attenuated in virulence in rodent assays, while mutants with minor defects in the mannan structure (e.g., mnn4D) tend to be unaltered in virulence and are able to stimulate significant levels of cytokine production. Conclusion

Candida albicans, a normal commensal of mucosal surfaces and the digestive tract, presently stands as the most frequent cause of opportunistic fungal infections in immunocompromised individuals. Many of the key virulence traits of this fungus involve the activity of glycosylated proteins in the cell wall and in the secretome. As a result, ongoing research in a number of laboratories is now focused on Candida glycobiology in order to identify essential events in its pathogenesis that can be targeted by fungal chemotherapy. These include the identification and characterization of enzymes involved in critical steps of glycoprotein assembly and secretion, as well as the role of O‑ and N‑linked glycans during recognition and adhesion to host cells and virulence. The combined use of biochemistry, molecular biology and physiology will hopefully lead to a better understanding of C. albicans biology and the possibility of preventing and/or treating disseminated forms of the disease. Future perspective

The incidence of candidiasis caused by C. albi‑ cans and other species of Candida has increased over the past decade. Intensive research in a number of laboratories all over the world using novel tools of biochemistry, molecular biology and physiology in combination with powerful technologies of glycoproteomics should provide future science group


additional and much-needed information for a deeper understanding of Candida glycobiology and pathogenesis within the next few years, and

Review

pave the way for the development of new therapeutic approaches for treating candidiasis and hopefully other mycoses.

Executive summary Candida albicans as a human pathogen: cell-wall components & their role in host-cell recognition C. albicans is the most common cause of opportunistic fungal infections in immunocompromised individuals. The mortality rate for systemic candidiasis is approximately 30–40%. n Cell-wall components, generically known as adhesins, play a determinant role in the interaction of the pathogen with the host cell. n Mutants with specific alterations in the cell wall have proved to be valuable tools to identify the surface components recognized by immune cells. n Cell-wall mannoproteins are recognized as major players in host–pathogen interaction and are therefore of indisputable importance in fungal pathogenesis. n n

Mannosyl & glucosyl transferases Mannose and glucose residues are added to acceptor proteins in the endoplasmic reticulum from dolichol-linked sugars, which are synthesized by dolichol phosphate mannose synthase (DPMS) and dolichol phosphate glucose synthase (DPGS), respectively. n Protein mannosylation is essential for many organisms. For instance, deficient mannosylation can result in severe forms of congenital disorders of glycosylation (CDGs). n DPMS is a potential antifungal target. Its biochemical properties and functional coupling to protein mannosyl transferases (PMTs) have been determined in membranes and solubilized fractions from C. albicans. These fractions also transfer glucose from radiolabeled UDP–Glc mainly to dolichol phosphate glucose. DPGS was identified and characterized for the first time in a pathogenic dimorphic fungus. n

N-glycan processing a-glucosidases The DP-linked Glc3M9 oligosaccharide is transferred to nascent polypeptides in the endoplasmic reticulum (ER) by the oligosaccharyl transfer complex (OST). n Shortly after transfer, Glc M is trimmed by processing a-glucosidases I and II and mannosidase I yielding M before glycoproteins exit 3 9 8 the ER or are targeted for degradation if they are misfolded. n A recombinant, 50-kDa a-glucosidase I was characterized in C. albicans. Also, a native a-glucosidase II was purified and separated into two polypeptides of 36 and 47 kDa, the latter being responsible for catalytic activity. n Most a-mannosidase activity (80%) in C. albicans is present in the soluble fraction in which two forms of the enzyme, named as a-1,2-mannosidases I and II, can be recognized. The remaining activity is associated to the ER membrane. n Cytosolic a-1,2-mannosidase I is produced by proteolysis of the membrane-bound enzyme by the Kex2 protease whereas a-1,2mannosidase II is an in vitro artifact generated by proteolysis during preparation of cell extracts. n All three forms of the enzyme exhibit the same catalytic properties and are immunorelated. n The presence of an a-1,2-mannosidase of family 47 in the cytosol of a lower eukaryote has been demonstrated. n

N- and O-linked mannans & the C. albicans–host interaction The study of mannosylation in a pathogen such as C. albicans is important to determine the role of mannans in virulence and fungal immune recognition. n A number of mannosylation mutants in C. albicans show defects in growth rates in vitro that strongly correlate with attenuation of virulence. n Mannosylation mutants also exhibit changes in cell-wall composition. n Disruptions of crucial steps of mannosylation can affect the folding, transport and activity of key virulence factors in the cell wall. Therefore, cell-wall homeostasis is essential for C. albicans viability and virulence. n

Immune recognition of C. albicans glycans b-glucans and mannans are pathogen-associated molecular patterns recognized by pattern-recognition receptors present in the surface of immune cells. n The mannose receptor (MR) is the main receptor for N-linked mannans during the immune recognition of C. albicans by macrophages and monocytes. n C. albicans recognition by dendritic cells requires a second receptor, the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing-nonintegrin (DC-SIGN). n Phospholipomannans may be also relevant to the interaction of C. albicans with the host immune system. n N-linked mannans structures on the hyphal surface may be different from those present on the yeast cell wall. n There seems to be a direct relationship between mannan structure, activation of immune response and virulence. n

Future perspective Research using novel tools of biochemistry, molecular biology and physiology in combination with glycoproteomics should provide much-needed information to understand Candida glycobiology within the next few years, and pave the way for the development of new therapeutic approaches for treating candidiasis and hopefully other mycoses.

n


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Presently, much of what we know of fungal glycobiology comes from studies carried out in S. cerevisiae, a nonpathogenic yeast phylogenetically distant from C. albicans. Much remains to be learned about the catalytic mechanism and regulation of enzymes and other factors involved in critical steps of glycan assembly and structure. In addition to considering more detailed studies of the glycosyltransferases and glycosidases covered in this review, the analysis of enzymes responsible for the synthesis of N-glycan outer chains and the role of these oligosaccharides in immune recognition and virulence remains an important area of biomedical research. Bibliography Papers of special note have been highlighted as: n of interest nn of considerable interest 1.

2.

3.

4.

5.

6.

7.

8.

Mathews HL, Witek-Janusek L: Host defense against oral, esophageal and gastro-intestinal candidiasis. In: Candida and Candidiasis. Calderone RA (Ed.). ASM Press, Washington, DC, 179–192 (2002). Romani L: Immunology of invasive candidiasis. In: Candida and Candidiasis. Calderone RA (Ed.). ASM Press, Washington, DC, 223–241 (2002). Blot S, Hoste E, Vandewoude K, Colardyn F: Estimates of attributable mortality of systemic Candida infection in the ICU. J. Crit. Care 18, 130–131 (2003). Bille J, Marchetti O, Calandra T: Changing face of health-care associated fungal infections. Curr. Opin. Infect. Dis. 18, 314–319 (2005). Sandini S, La Valle R, De Bernardis F, Macrì C, Cassone A: The 65 kDa mannoprotein gene of Candida albicans encodes a putative b‑glucanase adhesin required for hyphal morphogenesis and experimental pathogenicity. Cell. Microbiol. 9, 1223–1238 (2007). Ruiz-Herrera J, Elorza MV, Valentín E, Sentandreu R: Molecular organization of the cell wall of Candida albicans and its relation to pathogenicity. FEMS Yeast Res. 6, 14–29 (2006). Dranginis AM, Rauceo JM, Coronado JE, Lipke PN: A biochemical guide to yeast adhesins: glycoproteins for social and antisocial occasions. Microbiol. Mol. Biol. Rev. 71, 282–294 (2007). Butler G, Rasmussen MD, Lin FM et al.: Evolution of pathogenicity and sexual reproduction in eight Candida genomes. Nature 459, 657–662 (2009).

1178

9.

Acknowledgements

The authors are indebted to Claudia Iris Robledo-Ortiz for her valuable help with the figures, and other people, mostly students, who directly or indirectly contributed to the work presented in this review. Financial & competing interests disclosure

Research in the laboratories of Patricia Ponce-Noyola, Arturo Flores-Carreón and Everardo López-Romero was jointly supported by grants Nos. 2005-C0-11919, 83414, 458100-5-0041PN from CONACyT, México, and 39528-Q from SEP-CONACyT, México. Research in the laboratory of Neil AR Gow and Héctor M MoraMontes was supported by grant No. 080088 from The Wellcome Trust, London, UK.

Kamai Y, Kubota M, Kamai Y, Hosokawa T, Fukuoka T, Filler SG: Contribution of Candida albicans ALS1 to the pathogenesis of experimental oropharyngeal candidiasis. Infect. Immun. 70, 5256–5258 (2002).

and PRRs interplay leading to tolerance or infection by Candida albicans. Cell. Microbiol. 11(7), 1007 (2009). 18. Prill SK-H, Klinkert B, Timpel C, Gale CA,

Schröppel K, Ernst JF: PMT family of Candida albicans: five protein mannosyltransferase isoforms affect growth, morphogenesis and antifungal resistance. Mol. Microbiol. 55, 546–560 (2005).

10. Oh SH, Cheng G, Nuessen JA et al.:

Functional specificity of Candida albicans Als3p proteins and clade specificity of ALS3 alleles discriminated by the number of copies of the tandem repeat sequence in the central domain. Microbiology 15, 673–681 (2005).

19. Willger SD, Ernst JF, Aspaugh JA,

Lengeler KN: Characterization of the PMT gene family in Cryptococcus neoformans. PLoS ONE 4, e6321 (2009).

11. Kumamoto CA, Vinces MD: Alternative

Candida albicans lifestyles: growth on surfaces. Annu. Rev. Microbiol. 59, 113–133 (2005).

20. Wheeler RT, Fink GR: A drug-sensitive

genetic network mask fungi from the immune system. PLoS Pathog. 2(4), e35 (2006).

12. Nobile CJ, Andes DR, Nett JE et al.: Critical

role of Bcr1-dependent adhesins in C. albicans biofilm formation in vitro and in vivo. PLoS Pathog. 2, e63 (2006).

21. Quing YY, de Groot PWJ, de Koster CG,

Klis FM: Mass spectrometry-based proteomics of fungal wall glycoproteins. Trends Microbiol. 16, 20–26 (2008).

13. Nobile CJ, Mitchell AP: Genetics and genomics

of Candida albicans biofilm formation. Cell. Microbiol. 8, 1382–1391 (2006).

22. Lengeler KB, Tielker D, Ernst JF: Protein-O-

mannosyltransferases in virulence and development. Cell. Mol. Life Sci. 65, 528–544 (2008).

14. Sheppard DC, Yeaman MR, Welch WH

et al.: Functional and structural diversity in the Als protein family of Candida albicans. J. Biol. Chem. 279, 30480–30489 (2004). 15. Zhao X, Daniels KJ, Oh SH et al.: Candida

albicans Als3p is required for wild-type biofilm formation on silicone elastomer surfaces. Microbiology 152, 2287–2299 (2006). 16. Staab JF, Bahn YS, Tai CH, Cook PF,

Sundstrom P: Expression of transglutaminase substrate activity on Candida albicans germ tubes through a coiled, disulfide-bonded N‑terminal domain of Hwp1 requires C‑terminal glycosylphosphatidylinositol modification. J. Biol. Chem. 279, 40737–40747 (2004). 17. Jouault T, Sarazin A, Martinez-Esparza M,

Fradin C, Sendid B, Poulain D: Host responses to a versatile commensal: PAMPs


n

A recent account of the role of protein mannosyl transferases (PMTs) in virulence and development

23. Lommel M, Strahl S: Protein

O‑mannosylation: conserved from bacteria to humans. Glycobiology 19, 816–828 (2009). nn

An excellent, expert and recent review on O-linked mannosylation in a wide number of divergent organisms

24. Nunes LR, Costa de Oliveira R, Batista DL

et al.: Transcriptome analysis of Paracoccidioides brasiliensis cells undergoing mycelium-to-yeast transition. Eukaryot. Cell 4, 2115–2128 (2005). 25. Herscovics A, Orleans P: Glycoprotein

biosynthesis in yeast. FASEB J. 7, 540–550 (1993).



26. Deshpande N, Wilkins M, Packer N,

Nevalainen H: Protein glycosylation pathways in filamentous fungi. Glycobiology 18, 626–637 (2008). n

38. Strahl-Bolsinger S, Gentzsch M, Tanner W:

39. Abeijon C, Yanagisawa K, Mandon E et al.:

Guanosine diphosphatase is required for protein and sphingolipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae. J. Cell Biol. 122, 307–323 (1993).

27. Hofsteenge J, Löfler A, Müller DR,

Richter WJ, de Beer T, Vliegenthart JFG: Techniques in protein chemistry VII. In: Protein C-glycosylation. Marshak DR (Ed.). Academic Press, NY, USA, 163–171 (1996).

Domínguez A, Abeijon C: The Golgi GDPase of the fungal pathogen Candida albicans affects morphogenesis, glycosylation, and cell wall properties. Eukaryot. Cell 1, 420–431 (2002).

30. Leroy JG: Congenital disorders of

Dolichol phosphate mannose synthase (DPM1) mutations define congenital disorder of glycosylation 1e (CDG‑1e). J. Clin. Invest. 105, 191–198 (2000). n

synthase is required in vivo for glycosyl phosphatidylinositol membrane anchoring, O mannosylation, and N glycosylation of protein in Saccharomyces cerevisiae. Mol. Cell. Biol. 10, 5796–5805 (1990). nn

Demonstrates for the first time that dolichol phosphate mannose synthase (DPMS) is an essential protein.

34. Leidich SD, Drapp DA, Orlean P: A

conditionally lethal yeast mutant blocked at the first step of glycosyl phosphatidylinositol anchor synthesis. J. Biol. Chem. 269, 10193–10196 (1994). 35. Nagamune K, Nozaki T, Maeda Y et al.:

Critical roles of glycosylphosphatidylinositol for Trypanosoma brucei. Proc. Natl Acad. Sci. USA 97, 10336–10341 (2000). 36. Abeijon C, Hirschberg CB: Topography of

initiation of N‑glycosylation reactions. J. Biol. Chem. 265, 14691–14695 (1990). 37. Gemmill TR, Trimble RB: Overview of

N‑ and O‑linked oligosaccharide structures found in various yeast species. Biochim. Biophys. Acta 1426, 227–237 (1999).


51. Lamani E, Mewbourne RB, Fletcher DS

et al.: Structural studies and mechanism of Saccharomyces cerevisiae dolichylphosphate‑mannose synthase: insights into the initial step of synthesis of dolichylphosphate‑linked oligosaccharide chains in membranes of endoplasmic reticulum. Glycobiology 16, 666–678 (2006). 52. Maeda Y, Tomita S, Watanabe R, Ohishi K,

Kinoshita T: DPM2 regulates biosynthesis of dolichol phosphate‑mannose in mammalian cells: correct subcellular localization and stabilization of DPM1, and binding of dolichol phosphate. EMBO J. 17, 4920–4929 (1998).

43. Maeda Y, Kinoshita T: Dolichol-phosphate

mannose synthase: structure, function and regulation. Biochim. Biophys. Acta 1780, 861–868 (2008).

32. Nozaki M, Ohishi K, Yamada N,

33. Orlean P: Dolichol phosphate mannose

Orlean P, Penttila M, Palamarczyk G: Dolichol phosphate mannose synthase from the filamentous fungus Trichoderma reesei belongs to the human and Schizosaccharomyces pombe class of the enzyme. Glycobiology 10, 983–991 (2000).

Demonstrates that alterations in mannosylation can result in congenital disorders of glycosylation. Deficiency of dolichol-phosphate‑mannose synthase‑1 causes congenital disorder of glycosylation type 1e. J. Clin. Invest. 105, 233–239 (2000).

31. Freeze HH: Congenital disorders of

Kinoshita T, Nagy A, Takeda J: Developmental abnormalities of glycosylphosphatidylinositol-anchor-deficient embryos revealed by Cre/loxP system. Lab. Invest. 79, 293–299 (1999).

50. Kruszewska S, Saloheimo M, Migdalski A,

42. Imbach T, Schenck V, Schollen E et al.:

N‑glycosylation including diseases associated with O‑ as well as N‑glycosylation defects. Pediatr. Res. 60, 643–656 (2006). glycosylation: CDG‑I, CDG‑II, and beyond. Curr. Mol. Med. 7, 389–396 (2007).

McConville RJ: Evidence that free GPI glycolipids are essential for growth of Leishmania mexicana. EMBO J. 18, 2746–2755 (1999).

41. Kim S, Westphal V, Srikrishna G et al.:

29. Lehle L, Strahl S, Tanner W: Protein

glycosylation, conserved from yeast to man: a model organism helps elucidate congenital human diseases. Angew. Chem. Int. Ed. Engl. 45, 6802–6818 (2006).

49. llgoutz SC, Zawadski, JL, Ralton JE,

40. Herrero AB, Uccelletti D, Hirschberg CB,

28. de Beer T, Vliegenhart JF, Löffler A,

Hofsteenge J: The hexopyranosyl residue that is glycosidically linked to the side chain of tryptophan‑7 in human RNase Us is a‑mannopyranose. Biochemistry 34, 11785–11789 (1995).

sufficient for synthesis of dolicholphosphate‑mannose in mammalian cells. J. Biol. Chem. 273, 9249–9254 (1998).

Protein O‑mannosylation. Biochim. Biophys. Acta 1426, 297–307 (1999).

A recent, short review of glycoprotein biosynthesis in fungi.

nn

An important contribution to the detailed knowledge of DPMS with emphasis on the human enzyme.

53. Ashida H, Maeda Y, Kinoshita T: DPM1, the

catalytic subunit of dolichol-phosphate mannose synthase, is tethered to and stabilized on the endoplasmic reticulum membrane by DPM3. J. Biol. Chem. 281, 896–904 (2006).

44. Orlean P, Albright C, Robbins PW: Cloning

and sequencing of the yeast gene for dolichol phosphate mannose synthase, an essential protein. J. Biol. Chem. 263, 17499–17507 (1988).

54. Arroyo-Flores BL, Calvo-Méndez C,

Flores-Carreón A, López-Romero E: Biosynthesis of glycoproteins in Candida albicans: activity of dolichol phosphate mannose synthase and protein mannosylation in a mixed membrane fraction. Microbiology 141, 2289–2294 (1995).

45. Mazhari T, Eckert B, Blank M et al.: Cloning

and functional expression of glycosyltransferases from parasitic protozoans by heterologous complementation in yeast: the dolichol phosphate mannose synthase from Trypanosoma brucei brucei. Biochem. J. 316, 853–858 (1996). 46. Zimmerman JW, Specht CA, Cazares BX,

Robbins PW: The isolation of Dol‑P‑Man synthase from Ustilago maydis that functions in Saccharomyces cerevisiae. Yeast 12, 765–771 (1996). 47. Colussi P, Taron CH, Mack JC, Orlean P:

Human and Saccharomyces cerevisiae dolichol phosphate mannose synthases represent two classes of the enzyme, but both function in Schizosaccharomyces pombe. Proc. Natl Acad. Sci. USA 94, 7873–7878 (1997). 48. Tomita S, Inoue N, Maeda Y, Ohishi K,

Takeda J, Kinoshita T: A homologue of Saccharomyces cerevisiae Dpm1p is not


Review

n

Demonstrates for the first time the activity of DPMS and its functional coupling with PMTs in Candida albicans.

55. Helenius A, Aebi M: Intracellular functions

of N‑linked glycans. Science 291, 2364–2369 (2001). 56. Spiro RG: Protein glycosylation: nature,

distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology 12, 43R–56R (2002). 57. Timpel C, Strahl-Bolsinger S,

Ziegelbauer K, Ernst JF: Multiple functions of Pmt1p-mediated protein O‑mannosylation in the fungal pathogen Candida albicans. J. Biol. Chem. 273, 20837–20846 (1998).

1179

Review


58. Timpel C, Zink S, Strahl-Bolsinger S,

67. Arroyo-Flores BL, Calvo-Méndez C,

Schroppel K, Ernst JF: Morphogenesis, adhesive properties, and antifungal resistance depend on the Pmt6 protein mannossyltransferase in the fungal pathogen Candida albicans. J. Bacteriol. 182, 3063–3071 (2000). 59. Arroyo-Flores BL, Calvo-Méndez C,

Flores-Carreón A, López-Romero E: Biosynthesis of glycoproteins in Candida albicans: solubilization and partial characterization of dolichol phosphate mannose synthase and protein mannosyl transferases. Ant. van Leeuw. 73, 289–297 (1998). 60. Arroyo-Flores BL, Rodríguez-Bonilla J,

Villagómez-Castro JC, Calvo-Méndez C, Flores-Carreón A, López-Romero E: Biosynthesis of glycoproteins in Candida albicans: activity of mannosyl and glucosyl transferases. Fungal Gen. Biol. 30, 127–133 (2000). 61. López-Romero E, Flores-Carreón A,

Arroyo-Flores BL et al.: Glycosyl transferases and glycosidases of glycoprotein biosynthesis with emphasis on Candida albicans and Entamoeba histolytica. Rec. Res. Devel. Microbiol. 4, 667–681 (2000). 62. Arroyo-Flores BL, Calvo-Méndez C,

Flores-Carreón A, López-Romero E: Biosynthesis of glycoproteins in the pathogenic fungus Candida albicans: activation of dolichol phosphate mannose synthase by cAMP-mediated protein phosphorylation. FEMS Immunol. Med. Microbiol. 45, 429–434 (2005). nn

Describes the purification of DPMS from C. albicans and the in vitro regulation of the enzyme by protein phosphorylation.

63. Kruszewska JS, Perlinska-Lenart U,

Palamarczyk G: Solubilization and one-step purification of mannosylphosphoryldolichol synthase from Trichoderma reesei. Acta Biochim. Pol. 43, 397–401 (1996). 64. Villagómez-Castro JC, Calvo-Méndez C,

Flores-Carreón A, López-Romero E: Partial purification and characterization of dolichol phosphate mannose synthase from Entamoeba histolytica. Glycobiology 10, 1311–1316 (2000). 65. Banerjee DK, Kousvelari EE, Baum BJ:

cAMP-mediated protein phosphorylation of microsomal membranes increases mannosylphospho-dolichol synthase activity. Proc. Natl Acad. Sci. USA 84, 6389–6393 (1987). 66. Banerjee DK, DaSilva JJ, Bigio B:

Mannosyl-phosphodolichol synthase activity is associated with a 32 kDa phosphoprotein. Biosci. Rep. 19, 169–177 (1999).

1180

Flores-Carreón A, López-Romero E: Partial purification and characterization of a mannosyl transferase involved in O‑linked mannosylation of glycoproteins in Candida albicans. Ant. van Leeuw. 85, 199–207 (2004). n

Describes the partial purification and characterization of a PMT in C. albicans.

n

77. Gold P, Green M: Partial purification and

properties of a murine plasmacytoma glucosyltransferase. J. Biol. Chem. 258, 12967–12975 (1983). 78. Scher MG, Jochen A, Waechter CJ:

68. Kornfeld R, Kornfeld S: Assembly of

Biosynthesis of glucosylated derivatives of dolichol: possible intermediates in the assembly of white matter glycoproteins. Biochemistry 16, 5037–5044 (1977).

asparagine‑linked oligosaccharides. Ann. Rev. Biochem. 54, 631–664 (1985). 69. Ballou L, Gopal P, Krummel B, Tammi M,

Ballou CE: A mutation that prevents glucosylation of the lipid‑linked oligosaccharide precursor leads to underglycosylation of secreted yeast invertase. Proc. Natl Acad. Sci. USA 83, 3081–3085 (1986).

79. Forsee WT, Elbein AD: Biosynthesis of

mannosyl- and glucosyl-phosphorylpolyprenols in cotton fibers. J. Biol. Chem. 248, 2858–2867 (1973). 80. Keenan RW, Matula JM, Holloman L:

Studies on the biosynthesis of glucolipid in Tetrahymena pyriformis. Biochim. Biophys. Acta 326, 84–92 (1973).

70. Heesen S, Lehle L, Weissmann A, Aebi M:

Isolation of the ALG5 locus encoding the UDP-glucose:dolichyl-phosphate glucosyltransferase from Saccharomyces cerevisiae. Eur. J. Biochem. 224, 71–79 (1994).

81. Banerjee DK: Amphomycin inhibits

mannosyl-phosphoryldolichol synthesis by forming a complex with dolichylmonophosphate. J. Biol. Chem. 264, 2024–2028 (1989).

71. Runge KW, Huffaker TC, Robbins PW:

Two yeast mutations in glucosylation steps of the asparagine glycosylation pathway. J. Biol. Chem. 259, 412–417 (1984). 72. Muñoz MD, Hernández LM, Basco R,

Andaluz E, Larriba G: Glycosylation of yeast exoglucanase sequons in alg mutants deficient in the glucosylation steps of the lipid‑linked oligosaccharide. Presence of glucotriose unit in Dol‑PP‑GlcNAc2Man9Glc3 influences both glycosylation efficiency and selection of N‑linked sites. Biochim. Biophys. Acta 1201, 361–366 (1994). 73. Palamarczyk G, Drake R, Haley B,

Lennarz WJ: Evidence that the synthesis of glucosylphosphodolichol in yeast involves a 35‑kDa membrane protein. Proc. Natl Acad. Sci. USA 87, 2666–2670 (1990). 74. Drake RR, Palamarczyk G, Haley BE,

Lennarz WJ: Evidence for the involvement of a 35‑kDa membrane protein in the synthesis of glucosylphosphoryldolichol. Biosci. Rep. 10, 61–68 (1990). 75. Drake RR, Kaushal GP, Pastuszak I,

Elbein AD: Partial purification, photoaffinity labeling, and properties of mung bean UDP-glucose:dolichylphosphate glucosyltransferase. Plant Physiol. 97, 396–401 (1991). 76. Rodríguez-Bonilla J, Vargas-Rodríguez L,

Calvo-Méndez C, Flores-Carreón A, López-Romero E: Biosynthesis of glycoproteins in Candida albicans: biochemical characterization of dolichol phosphate glucose synthase. Ant. van Leeuw. 73, 373–380 (1998). Future Microbiol. (2009) 4(9)

Describes the biochemical characterization of dolichol phosphate glucose synthase (DPGS) in C. albicans and demonstrates that UMP reverses the DPGS reaction.

82. Kelleher DJ, Gilmore R: An evolving view of

the eukaryotic oligosaccharyltransferase. Glycobiology 16, 47R–62R (2006). 83. Jones T, Federspiel NA, Chibana H et al.: The

diploid genome sequence of Candida albicans. Proc. Natl Acad. Sci. USA 111, 7329–7334 (2004). 84. Moremen KW, Trimble RB, Herscovics A:

Glycosidases of the asparagine‑linked oligosaccharide processing pathway. Glycobiology 4, 113–125 (1994). 85. Herscovics A: Processing glycosidases of

Saccharomyces cerevisiae. Biochim. Biophys. Acta 1426, 275–285 (1999). n

An old but frequently cited review on processing a-glycosidases in Saccharomyces cerevisiae.

86. Herscovics A: Importance of glycosidases in

mammalian glycoprotein biosynthesis. Biochim. Biophys. Acta 1473, 96–107 (1999). 87. Shailubhai K, Pukazhenthi BS, Saxena FS,

Varma GM, Vijay IK: Glucosidase I, a transmembrane endoplasmic reticular glycoprotein with a luminal catalytic domain. J. Biol. Chem. 266, 16587–16593 (1991). 88. Yet MG, Shao MC, Wold F: Effects of the

protein matrix on glycan processing in glycoproteins. FASEB J. 2, 22–31 (1988). 89. Byrd JC, Tarentino AL, Maley F, Atkinson PH,

Trimble RB: Glycoprotein synthesis in yeast. Identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing. J. Biol. Chem. 257, 14657–14666 (1982).



90. Henrissat B, Davis G: Structural and

nn

sequence-based classification of glycoside hydrolases. Curr. Opin. Struct. Biol. 7, 637–644 (1997). 91. Kilker RD, Saunier B, Tkacz JS, Herscovics A:

Partial purification from Saccharomyces cerevisiae of a soluble glucosidase which removes the terminal glucose from the oligosaccharide Glc3Man9GlcNAc2. J. Biol. Chem. 256, 5299–5303 (1981). 92. Romero PA, Dijkgraaf GJ, Shahinian S,

Herscovics A, Bussey H: The yeast CWH41 gene encodes glucosidase I. Glycobiology 7, 997–1004 (1997). 93. Kalz-Fuller B, Bieberich E, Bause E: Cloning

and expression of glucosidase I from human hippocampus. Eur. J. Biochem. 231, 344–351 (1995).

Endoplasmic reticulum glucosidase II is composed of a catalytic subunit, conserved from yeast to mammals, and a tightly bound no catalytic HDEL‑containing subunit. J. Biol. Chem. 271, 27509–27516 (1996). Quaternary and domain structure of glycoprotein processing glycosidase II. Biochemistry 40, 10717–10722 (2001). Parodi AJ: Genetic evidence for the heterodimeric structure of glucosidase II. The effect of disrupting the subunit-encoding genes on glycoprotein folding. J. Biol. Chem. 274, 25899–25905 (1999). The a‑ and b‑subunits are required for expression of catalytic activity in the hetero-dimeric glucosidase II complex from human liver. Glycobiology 10, 493–502 (2000). than one glycan is needed for ER glycosidase II to allow entry of glycoproteins into the calnexin/calreticulin cycle. Mol. Cell 19, 183–195 (2005). Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure. Cell Biol. 142, 1223–1233 (1998). GTB1 encodes a subunit of glucosidase II required for glycoprotein processing in the endoplasmic reticulum. J. Biol. Chem. 281, 6325–6333 (2006).

102. Mora-Montes HM, Bates S, Netea MG et al.:

Endoplasmic reticulum a‑glycosidases of Candida albicans are required for N glycosylation, cell wall integrity, and normal host–fungus interaction. Eukaryot. Cell 6, 2184–2193 (2007).


glycoprotein quality control in the secretory pathway. Trends Biochem. Sci. 26, 619–624 (2001). 117. Dennis JW, Warren CE, Granovsky M,

Demetriou M: Genetic defect in the N‑glycosylation and cellular diversity in mammals. Curr. Opin. Struct. Biol. 11, 601–607 (2001). 118. Vázquez-Reyna AB, Balcázar-Orozco R,

Flores-Carreón A: Biosynthesis of glycoproteins in Candida albicans: biochemical characterization of a soluble a‑mannosidase. FEMS Microbiol. Lett. 106, 321–326 (1993). 119. Vázquez-Reyna AB, Ponce-Noyola P,

Calvo-Mèndez C, Lòpez-Romero E, Flores-Carreón A: Purification and biochemical characterization of two soluble a‑mannosidases from Candida albicans. Glycobiology 9, 533–537 (1999). 120. Vázquez-Reyna AB, Balcázar-Orozco R,

Calvo-Mèndez C, Lòpez-Romero E, Flores-Carreón A: Processing of the Man10GlcNAc (M10) oligosaccharide by a‑mannosidases from Candida albicans. FEMS Microbiol. Lett. 85, 37–41 (2000).

110. Torre-Bouscoulet ME, López-Romero E,

Balcázar-Orozco R, Calvo-Méndez C, Flores-Carreón A: Partial purification and biochemical characterization of a soluble a‑glucosidase II-like activity from Candida albicans. FEMS Microbiol. Lett. 236, 123–128 (2004).

100. Faridmoayer A, Scaman CH: Binding residues

functional carboxylic acid residues of yeast processing a‑glucosidase I. Glycoconj. J. 24, 429–437 (2007).

116. Cabral CM, Liu Y, Sifer RN: Dissecting

109. Wilkinson BM, Purswani J, Stirling CJ: Yeast

relationships in glucosidase I: amino acids involved in binding the substrate to the enzyme. Glycobiology 7, 399–404 (1997).

101. Faridmoayer A, Scaman CH: Truncation and

of a cDNA encoding a novel human Golgi a1,2‑mannosidase (IC) involved in N-glycan biosynthesis. J. Biol. Chem. 271, 31655–31660 (2000).

108. Jacob CA, Burda P, Roth J, Aebi MJ:

99. Romaniouk A, Vijay AK: Structure–function

and catalytic domain of soluble Saccharomyces cerevisiae processing a‑glucosidase I. Glycobiology 15, 1341–1348 (2005).

115. Tremblay LO, Herscovics A: Characterization

107. Deprez P, Gautschi M, Helenius A: More

98. Pukazhenthi BS, Muniappa N, Vijay IK: Role

of sulfhydryl groups in the function of glucosidase I from mammary gland. J. Biol. Chem. 268, 6445–6452 (1993).

Herscovics A, Moremen KW: Substrate specificities of recombinant murine Golgi a1,2‑mannosidases IA and IB and comparison with endoplasmic reticulum and Golgi processing a1,2‑mannosidases. Glycobiology 8, 981–995 (1998).

106. Treml K, Meimaroglou D, Hentges A, Bause E:

97. Dhanawansa R, Faridmoayer A,

van der Merwe G, Li YX, Scaman CH: Overexpression, purification, and partial characterization of Saccharomyces cerevisiae processing a‑glucosidase I. Glycobiology 12, 229–234 (2002).

114. Lal A, Pang P, Kalelkar S, Romero PA,

105. D’Alessio C, Fernandez F, Trombetta ES,

96. Faridmoayer A, Scaman CH: An improved

purification procedure for soluble processing a‑glucosidase I from Saccharomyces cerevisiae overexpressing CWH41. Protein Expr. Purif. 33, 11–18 (2004).

expression of a specific human a1,2‑mannosidase that trims Man9GlcNAc2 to Man8GlcNAc2 isomer B during N-glycan biosynthesis. Glycobiology 9, 1073–1078 (1999).

104. Trombetta ES, Fleming KG, Helenius A:

95. Bause E, Schweden J, Gross A, Orthen B:

Purification and characterization of trimming glucosidase I from pig liver. Eur. J. Biochem. 183, 661–669 (1989).

113. Tremblay LO, Herscovics A: Cloning and

103. Trombetta ES, Simons JF, Helenius A:

94. Jiang B, Sheraton J, Ram AF, Dijkgraaf GJ,

Klis FM, Bussey H: CWH41 encodes a novel endoplasmic reticulum membrane N-glycoprotein involved in b,1,6‑glucan assembly. J. Bacteriol. 178, 1162–1171 (1996).

A key contribution to understanding the role of endoplasmic reticulum a-glycosidases in C. albicans glycobiology and its interaction with host cells.

n

121. Mora-Montes HM, López-Romero E,

Zinker S, Ponce-Noyola P, Flores-Carreòn A: Hydrolysis of Man9GlcNAc2 and Man8GlcNAc2 oligosaccharides by a purified a‑mannosidase from Candida albicans. Glycobiology 14, 593–598 (2004).

Describes for the first time the purification and characterization of a-glucosidase II from C. albicans.

111. Bourne Y, Henrissat B: Glycoside hydrolases

and glycosyltransferases: families and functional modules. Curr. Opin. Sruct. Biol. 11, 593–600 (2001). 112. Jelinek-Kelly S, Akiyama T, Saunier B, Tkacz

JS, Herscovics A: Characterization of specific a‑mannosidase involved in oligosaccharide processing in Saccharomyces cerevisiae. J. Biol. Chem. 260, 2253–2257 (1985).


Review

n

Describes the purification of a soluble a-mannosidase from C. albicans and its ability to trim natural oligosaccharides.

122. Jelinek-Kelly S, Herscovics A: Glycoprotein

biosynthesis in Saccharomyces cerevisiae: purification of the a‑mannosidase which removes one specific mannose residue from Man9GlcNAc. J. Biol. Chem. 263, 4757–4763 (1988).

1181

Review


123. Sentandreu M, Elorza MV, Sentandreu R,

Fonzi WA: Cloning and characterization of PRA1, a gene encoding a novel pH-regulated antigen of Candida albicans. J. Bacteriol. 180, 282–289 (1998). 124. Fonzi WA, Irwin MV: Isogenic strain

construction and gene mapping in Candida albicans. Genetics 134, 717–728 (1993). 125. Mora-Montes HM, Bader O,

López-Romero E et al.: Kex2 protease converts the endoplasmic reticulum a1,2‑mannosidase of Candida albicans into a soluble cytosolic form. Microbiology 154, 3782–3794 (2008). nn

A key paper to understand the precursorproduct relationship between the endoplasmic reticulum membrane-bound and soluble a-mannosidases in C. albicans.

126. Wu Y, Termine DJ, Swulius MT,

Moremen KW, Sifers RN: Human endoplasmic reticulum mannosidase I is subject to regulated proteolysis. J. Biol. Chem. 282, 4841–4849 (2007). 127. Mora-Montes HM, López-Romero E,

Zinker S, Ponce-Noyola P, Flores-Carreón A: Heterologous expression and biochemical characterization of an a1,2‑mannosidase encoded by the Candida albicans MNS1 gene. Mem. Inst. Oswaldo Cruz 103, 724–730 (2008). 128. Sanchez-Martinez CJ, Perez-Martin:

Dimorphism in fungal pathogens: Candida albicans and Ustilago maydis – similar inputs, different outputs. Curr. Opin. Microbiol. 4, 214–221 (2001). 129. Rouabhia M, Schaller M, Corbucci C et al.:

Virulence of the fungal pathogen Candida albicans requires the five isoforms of protein mannosyltransferases. Infect. Immun. 73, 4571–4580 (2005). 130. Bates S, Hughes HB, Munro CA et al.: Outer

chain N‑glycans are required for cell wall integrity and virulence of Candida albicans. J. Biol. Chem. 281, 90–98 (2006). nn

A key paper dealing with the relationship between outer chain N-glycans, cell-wall structure and virulence in C. albicans.

131. Bai C, Xu XL, Chan FY, Lee RT,

Wang Y: MNN5 encodes an iron-regulated a‑1,2‑mannosyltransferase important for protein glycosylation, cell wall integrity, morphogenesis, and virulence in Candida albicans. Eukaryot. Cell 5, 238–247 (2006). 132. Buurman ET, Westwater C, Hube B,

Brown AJ, Odds FC, Gow NA: Molecular analysis of CaMnt1p, a mannosyl transferase important for adhesion and virulence of Candida albicans. Proc. Natl Acad. Sci. USA 95, 7670–7675 (1998).

1182

133. Munro CA, Bates S, Buurman ET et al.:

Mnt1p and Mnt2p of Candida albicans are partially redundant a‑1,2‑mannosyltransferases that participate in O‑linked mannosylation and are required for adhesion and virulence. J. Biol. Chem. 280, 1051–1060 (2005). 134. Bates S, MacCallum DM, Bertram G et al.:

Candida albicans Pmr1p, a secretory pathway P‑type Ca 2+/Mn 2+ ‑ATPase, is required for glycosylation and virulence. J. Biol. Chem. 280, 23408–23415 (2005). 135. Southard SB, Specht CA, Mishra C,

Chen-Weiner J, Robbins PW: Molecular analysis of the Candida albicans homolog of Saccharomyces cerevisiae MNN9, required for glycosylation of cell wall mannoproteins. J. Bacteriol. 181, 7439–7448 (1999). 136. Warit S, Zhang N, Short A, Walmsley RM,

Oliver SG, Stateva LI: Glycosylation deficiency phenotypes resulting from depletion of GDP‑mannose pyrophosphorylase in two yeast species. Mol. Microbiol. 36, 1156–1166 (2000). 137. Nishikawa A, Poster JB, Jigami Y, Dean N:

Molecular and phenotypic analysis of CaVRG4, encoding an essential Golgi apparatus GDP‑mannose transporter. J. Bacteriol. 184, 29–42 (2002). 138. Rieg, G, Fu Y, Ibrahim AS, Zhou X,

Filler SG, Edwards JE Jr: Unanticipated heterogeneity in growth rate and virulence among Candida albicans AAF1 null mutants. Infect. Immun. 67, 3193–3198 (1999). 139. Hobson RP, Munro CA, Bates S et al.: Loss of

cell wall mannosylphosphate in Candida albicans does not influence macrophage recognition. J. Biol. Chem. 279, 39628–39635 (2004). 140. Li RK, Cutler JE: Chemical definition of an

epitope/adhesin molecule on Candida albicans. J. Biol. Chem. 268, 18293–18299 (1993). 141. Sundstrom P: Adhesins in Candida albicans.

Curr. Opin. Microbiol. 2, 353–357 (1999). 142. Hoyer LL: The ALS gene family of Candida

albicans. Trends Microbiol. 9, 176–180 (2001). 143. Sundstrom P: Adhesion in Candida spp. Cell.

Microbiol. 4, 461–469 (2002). 144. Fradin C, Slomianny MC, Mille C et al.:

b‑1,2 oligomannose adhesin epitopes are widely distributed over the different families of Candida albicans cell wall mannoproteins and are associated through both N‑ and O‑glycosylation processes. Infect. Immun. 76, 4509–4517 (2008). 145. Pierce CG, Thomas DP, Lopez-Ribot JL:

Effect of tunicamycin on Candida albicans biofilm formation and maintenance. J. Antimicrob. Chemother. 63, 473–479 (2009).


146. Nather K, Munro CA: Generating cell surface

diversity in Candida albicans and other fungal pathogens. FEMS Microbiol. Lett. 285, 137–145 (2008). 147. Plaine A, Walker L, Da Costa G et al.:

Functional analysis of Candida albicans GPI-anchored proteins: roles in cell wall integrity and caspofungin sensitivity. Fungal Genet. Biol. 45, 1404–1414 (2008). 148. Grimme SJ, Colussi PA, Taron CH, Orlean P:

Deficiencies in the essential Smp3 mannosyltransferase block glycosylphosphatidylinositol assembly and lead to defects in growth and cell wall biogenesis in Candida albicans. Microbiology 150, 3115–3128 (2004). 149. Bulawa CE, Miller DW, Henry LK,

Becker JM: Attenuated virulence of chitin-deficient mutants of Candida albicans. Proc. Natl Acad. Sci. USA 92, 10570–10574 (1995). 150. Munro CA, Winter K, Buchan A et al.: Chs1

of Candida albicans is an essential chitin synthase required for synthesis of the septum and for cell integrity. Mol. Microbiol. 39, 1414–1426 (2001). 151. Douglas CM, D’Ippolito JA, Shei GJ et al.:

Identification of the FKS1 gene of Candida albicans as the essential target of 1,3‑b‑d‑glucan synthase inhibitors. Antimicrob. Agents Chemother. 41, 2471–2479 (1997). 152. Herrero AB, Magnelli P, Mansour MK,

Levitz SM, Bussey H, Abeijon C: KRE5 gene null mutant strains of Candida albicans are avirulent and have altered cell wall composition and hypha formation properties. Eukaryot. Cell 3, 1423–1432 (2004). 153. Umeyama T, Kaneko A, Watanabe H et al.:

Deletion of the CaBIG1 gene reduces b‑1,6‑glucan synthesis, filamentation, adhesion, and virulence in Candida albicans. Infect. Immun. 74, 2373–2381 (2006). 154. Vecchiarelli A, Puliti M, Torosantucci A,

Cassone A, Bistoni F: In vitro production of tumor necrosis factor by murine splenic macrophages stimulated with mannoprotein constituents of Candida albicans cell wall. Cell. Immunol. 134, 65–76 (1991). 155. Netea MG, Van Der Graaf CA, Vonk AG,

Verschueren I, Van Der Meer JW, Kullberg BJ: The role of Toll-like receptor (TLR) 2 and TLR4 in the host defense against disseminated candidiasis. J. Infect. Dis. 185, 1483–1489 (2002). 156. Tada H, Nemoto E, Shimauchi H et al.:

Saccharomyces cerevisiae- and Candida albicans-derived mannan induced production of tumor necrosis factor‑a by human monocytes in a CD14- and Toll-like receptor 4-dependent manner. Microbiol. Immunol. 46, 503–512 (2002).



157. Brown GD, Herre J, Williams DL,

Willment JA, Marshall AS, Gordon S: Dectin‑1 mediates the biological effects of b‑glucans. J. Exp. Med. 197, 1119–1124 (2003). 158. Gantner BN, Simmons RM, Canavera SJ,

Akira S, Underhill DM: Collaborative induction of inflammatory responses by dectin‑1 and Toll-like receptor 2. J. Exp. Med. 197, 1107–1117 (2003). 159. Netea MG, Gow NA, Munro CA et al.:

Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and Toll-like receptors. J. Clin. Invest. 116, 1642–1650 (2006). 160. Gow NA, Netea MG, Munro CA et al.:

Immune recognition of Candida albicans b‑glucan by dectin-1. J. Infect. Dis. 196, 1565–1571 (2007). 161. Netea MG, Brown GD, Kullberg BJ, Gow NA:

An integrated model of the recognition of Candida albicans by the innate immune system. Nat. Rev. Microbiol. 6, 67–78 (2008). n

A recent account of the mechanism of recognition of C. albicans by the immune system.

162. Kery V, Krepinsky JJ, Warren CD, Capek P,

Stahl PD: Ligand recognition by purified human mannose receptor. Arch. Biochem. Biophys. 298, 49–55 (1992). 163. Newman SL, Holly A: Candida albicans is

phagocytosed, killed, and processed for antigen presentation by human dendritic cells. Infect. Immun. 69, 6813–6822 (2001). 164. Cambi A, Figdor CG: Dual function of C‑type

lectin-like receptors in the immune system. Curr. Opin. Cell Biol. 15, 539–546 (2003). 165. Cambi A, Gijzen K, de Vries JM et al.: The

C‑type lectin DC‑SIGN (CD209) is an antigen-uptake receptor for Candida albicans on dendritic cells. Eur. J. Immunol. 33, 532–538 (2003). 166. Cambi A, Netea MG, Mora-Montes HM et al.:

Dendritic cell interaction with Candida albicans critically depends on N‑linked mannan. J. Biol. Chem. 283, 20590–20599 (2008). 167. Shibata N, Arai M, Haga E et al.: Structural

identification of an epitope of antigenic factor 5 in mannans of Candida albicans NIH B‑792 (serotype B) and J‑1012 (serotype A) as b‑1,2‑linked oligomannosyl residues. Infect. Immun. 60, 4100–4110 (1992). 168. Jouault T, El Abed-El Behi M,

Martinez‑Esparza M et al.: Specific recognition of Candida albicans by


macrophages requires galectin‑3 to discriminate Saccharomyces cerevisiae and needs association with TLR2 for signaling. J. Immunol. 177, 4679–4687 (2006).

Website 201. Coutinho PM, Henrissat B: Carbohydrate-

active enzymes (1999). http://afmb.cnrs-mrs.fr/ ˜cazy/CAZY/index. html (Accessed May 2005)

169. Dalle F, Jouault T, Trinel PA et al.: b‑1,2‑ and

a‑1,2‑linked oligomannosides mediate adherence of Candida albicans blastospores to human enterocytes in vitro. Infect. Immun. 71, 7061–7068 (2003). 170. Fradin C, Slomianny MC, Mille C et al.:

Affiliations n

b‑1,2 oligomannose adhesin epitopes are widely distributed over the different families of Candida albicans cell wall mannoproteins and are associated through both N‑ and O‑glycosylation processes. Infect. Immun. 76, 4509–4517 (2008). 171. Han Y, Kanbe T, Cherniak R, Cutler JE:

n

Biochemical characterization of Candida albicans epitopes that can elicit protective and nonprotective antibodies. Infect. Immun. 65, 4100–4107 (1997). 172. Han Y, Morrison RP, Cutler JE: A vaccine

and monoclonal antibodies that enhance mouse resistance to Candida albicans vaginal infection. Infect. Immun. 66, 5771–5776 (1998).

n

173. Yamasaki S, Matsumoto M, Takeuchi O

et al.: C‑type lectin Mincle is an activating receptor for pathogenic fungus, Malassezia. Proc. Natl Acad. Sci. USA 106, 1897–1902 (2009). 174. Wells CA, Salvage-Jones JA, Li X et al.: The

macrophage‑inducible C‑type lectin, Mincle, is an essential component of the innate immune response to Candida albicans. J. Immunol. 180, 7404–7413 (2008).

n

175. Arizumi K, Shen GL, Shikano S et al.:

Cloning of a second dendritic cell-associated C‑type lectin (dectin-2) and its alternatively spliced isoforms. J. Biol. Chem. 275, 11957–11963 (2000).

n

176. Sato K, Yang XL, Yudate T et al.: Dectin‑2 is

a pattern recognition receptor for fungi that couples with the Fc receptorg chain to induce innate immune responses. J. Biol. Chem. 281, 38854–38866 (2006). 177. McGreal EP, Rosas M, Brown GD et al.: The

carbohydrate-recognition domain of dectin‑2 is a C‑type lectin with specificity for high mannose. Glycobiology 16, 422–430 (2006). 178. MacCallum DM, Castillo L, Brown AJ,

Gow NA, Odds FC: Early-expressed chemokines predict kidney immunopathology in experimental disseminated Candida albicans infections. PLoS One 4, e6420 (2009).


Review

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Héctor M Mora-Montes School of Medical Sciences, Institute of Medical Sciences, Foresterhill, University of Aberdeen, Aberdeen, AB25 2ZD, UK Tel.: +44 122 455 5879 Fax: +44 122 455 5844 [email protected] Patricia Ponce-Noyola Departamento de Biología, División de Ciencias Exactas y Naturales, Universidad de Guanajuato, Guanajuato, Gto. 36050, México Tel.: +52 473 732 0006 Fax: +52 473 732 0006 [email protected] Julio C Villagómez-Castro Departamento de Biología, División de Ciencias Exactas y Naturales, Universidad de Guanajuato, Guanajuato, Gto. 36050, México Tel.: +52 473 732 0006 Fax: +52 473 732 0006 [email protected] Neil AR Gow School of Medical Sciences, Institute of Medical Sciences, Foresterhill, University of Aberdeen, Aberdeen, AB25 2ZD, UK Tel.: +44 122 455 5879 Fax: +44 122 455 5844 [email protected] Arturo Flores-Carreón Departamento de Biología, División de Ciencias Exactas y Naturales, Universidad de Guanajuato, Guanajuato, Gto. 36050, México Tel.: +52 473 732 0006 Fax: +52 473 732 0006 [email protected] Everardo López-Romero Departamento de Biología, División de Ciencias Exactas y Naturales, Universidad de Guanajuato, Guanajuato, Gto. 36050, México Tel.: +52 473 732 0006 ext. 8156 Fax: +52 473 732 0006 [email protected]

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