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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
10.2217/FMB.09.88 © 2009
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
part of
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
Protein glycosylation in Candida
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
Review
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
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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
Protein glycosylation in Candida
Review
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
Review
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
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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
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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
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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
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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.
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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.
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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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