Macrosialin, a Mouse Macrophage-restricted Glycoprotein, Is a ...

Vol. 268,No. 13, Issue of May 5. pp. 9661-9666, 1993 Printed in U.S.A.

THE JOURNAL OF BIOLOGICALCHEMISTRY Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Macrosialin, a Mouse Macrophage-restricted Glycoprotein, Is a Member of the lamp/lgp Family* (Received for publication, August 20, 1992, and in revised form, September 10,1992)

Claire L. Holness, Rosangela P. da Silva$, Jonathan Fawcett, SiamonGordon$, and David L. Simmons8 From the Cell Adhesion Laboratory, Imperial Cancer Research Fund, Institute of Molecular Medicine, JohnRadcliffe Hospital, Headington, Oxford OX3 SDU, United Kingdom and the $Sir William Dunn Schoolof Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom

Macrosialin is a heavilyglycosylated transmembrane protein of 87-115 kDa, highly and specifically expressed bymouse tissue macrophages, and a tolesser extent by dendritic cells. We have isolatedcDNA clones a thioglycollate-elicited encodingmacrosialinfrom peritonealmacrophage cDNA library by transient expression in COS cells and panning with the antimacrosialin monoclonal antibodyFA11 1. A single 1.3kilobase macrosialin transcript was detected in both untreated and phorbol 12-myristate 13-acetate-stimulated RAW cells. The cDNA sequence predicts a type I integral membrane protein of 326 residues with a heavily glycosylated extracellular domainof 306 residues containing nine potential N-linked glycosylation sites and numerous potential 0-linked glycosylation sites. The extracellular domain consists of two distinct regions, separatedby an extended 12 residue prolinerich hinge; a membrane-distal mucin-like domain of 89 residues containing short peptide repeats andcona sisting of 44% serine and threonine residues; and membrane proximal domain of 170 residues, which has significant sequencehomology to a family of lysosomal associated glycoproteins known as the lamp-1 group.Macrosialin is the murine homologue of the human macrophage glycoprotein CD68 (72%identity, 80%similarity). Both proteins are preferentially expressed by macrophages and share the same bipartite structure having a mucin-like domain and a domain are common to the lampfamily. Macrosialin and CD68 the first examples of a lamp family protein with a restricted cell-type-specific expression. Theymay have evolved from the lamps to carry outspecialized functions in dedicated phagocyticcells.

Macrophages derive from the myeloid lineage and are believed to be the end-state of differentiation of blood monocytes. Macrophages are professional phagocytic cells and play key roles in many immune functions, including phagocytosis ~~~

*This workwas supported by the Medical Research Council (United Kingdom), The Wellcome Trust, and the Imperial Cancer Research Fund (United Kingdom). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequencefs)reported in thispaper has been submitted to the GenBankTM/EMBLDataBankwith accession number(s) X682 73. To whom all correspondence should be addressed ICRF Laboratories, Inst. of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom. Tel.: 44-865-222355;Fax: 44-865-222431.

of foreign and necrotic material and antigen processing and presentation (Gordon, 1986). Macrophages are resident in mostorgans and tissues butareparticularlyabundantin lymphoid organs (Gordon et al., 1992). Monoclonal antibodies specific for macrophages have been made bothforhuman (CD68) (Parwaresch et al., 1986; McMichael et al., 1987; Micklem et al., 1989; Warnke et al., 1989; Pulford et al., 1990; Knapp et al., 1991; Smith et al., 1991) and murine macrophages(FA11 antigen (Smith and Koch, 1987), now termed macrosialin (Rabinowitz and Gordon, 1989, 1991)). Macrosialin is the major sialylated glycoprotein of elicited macrophages and is differentially glycosylated in response to inflammatory stimuli. On SDS-PAGE,’ it resolves as a broadband of 87-115 kDa. Resident peritoneal macrophages express low levels of a non-lectin-binding glycoform, whereas inflammatory stimuli lead to a 17-fold increase in macrosialinexpression and acquisition of lectin binding properties (Rabinowitz and Gordon, 1991). The major remodeling occurs in 0-linked glycans with increase in sialylationandappearance of poly-N-acetyllactosamine structures. Macrosialin and CD68 share a number of common biochemical properties: macrophage-restricted expression, similar molecular mass (87-115 kDa), similar glycosylation patterns, being rich in both N-linked and especially 0-linked sugars and bearingnumerous terminal sialic acid residues, and mainly intracellular location in endosomes or lysosomes with only a small amount being expressed on the cell surface at any one time (Saito et al., 1991; Rabinowitz et. al., 1992). We have recently cloned a cDNA encoding CD68’ by transient expression in COS cells and found that it is a member of a family of lysosomal/endosomal associated glycoproteins known as the lamp/lgp family (human lamp-1, Fukuda et al. (1988); mouse lamp-1, Chen et al. (1988); mouse lamp-2, Cha et al. (1990); chicken lep100, Lippincot-Schwartz and Fambrough (1987), Fambrough et al. (1988) and Zot et al. (1990); and rat lgp120, Howe et al. (1988)). To better understand the structure and function of macrosialin, and more clearly establish the relationship of human CD68 to murine macrosialin, we report here the cloning of a cDNA clone encoding macrosialin by transient expression in COS cells and immunoselection (Seed and Aruffo, 1987) using the anti-macrosialin mAb FA/11 (Smith andKoch, 1987). Macrosialin is also a member of the lamp/lgp family and is the murine homologue of human The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; PMA, phorbol 12-myristate 13-acetate; mAb, monoclonal antibody; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s). * Holness, C. L., and Simmons, D. L. (1993) Blood, in press.

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CD68 (72% identity). Thus CD68/macrosialin are lineagerestricted, lysosomal-associated glycoproteins that could play roles in the specialized phagocytic activities of tissue macrophages, both in intracellularlysosomal metabolism and extracellular cell-cell and cell-pathogen interactions. MATERIALSANDMETHODS

Cell Lines and Culture Conditions-Thioglycollate-elicited peritoneal macrophages were obtained from BALB/c mice bred at the Sir William Dunn School of Pathology, 5 days after injection of 1.5 ml of Brewer's complete thioglycollate broth (Rabinowitz and Gordon, 1992). RAW cells were obtained from the Sir William Dunn School of Pathology, Oxford. All cells were grown in RPMI, 10% fetal calf serum (FCS). RAW cells were maintained a t a density of 5 X lo5ml" and induced with 25 ng ml" PMA for 24 h. The hybridoma secreting mAb FA/11, a rat IgGea, was maintained in Iscove's modified Dulbecco's medium (Flow Laboratories, Rickmansworth, United Kingdom) with 10% FCS, 0.1 mM hypoxanthine, 16 mM thymidine, glutamine, and antibiotics. The FA/11line was generously made available by Drs. M. Smith and G. Koch (Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom). Library Construction and Screening-A cDNA library was constructed in the expressionvectorpCDM8(Seed, 1987; Seed and Aruffo, 1987) from RNA prepared from thioglycollate-elicited peritonealmacrophages(BALB/c). Transfected COS cells transiently expressing macrosialin were isolated with the anti-macrosialin mAb FA/11 and pannedon plastic Petridishes coated with affinity-purified goat anti-rat IgG (Sigma). Episomal DNA was recovered from the panned cells, and the expression-panning cycle was repeated once more toobtain a cDNA clonedesignated pMS.l. Expression of macrosialin on the surface of transfected COS cells was detected by indirect immunofluorescence using mAb FA/11 and fluorescein-conjugated goat anti-rat IgG antibody (Sigma). Protein Labeling and Immunoprecipitations-J774 and RAW macrophage cell lines were grown in RPMI plus 10% FCS, 2 mM glutamine, penicillin, and streptomycin. COS cells and theB cell line NSO were grown in DMEM supplemented asabove. All cells were washed in methionine-free DMEM (Gibco Laboratories), preincubated in the same medium containing 10% dialyzed FCS for 1 h, after which 50 mCi/ml Tran%-label (ICN, Irving, CA) were added and cells incubated for another 16 h. Cells were then washed 3 times with cold phosphate-buffered saline and extracted with 2% octyl glucoside in 10 mM Tris, pH 8.0, plus protease inhibitors (5mM iodoacetamide, 2 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, 1 mg/ml pepstatin, and 1 mM leupeptin), for 1 h a t 4 "C. The lysates were spun for 5 min in a minicentrifuge and precleared with bovine IgG-coupled Sepharose. Macrosialin was then immunoprecipitated overnighta t 4 "C by mAb FA/11-coupled Sepharose. The beads were then washed in lysis buffer, boiled in SDS-Laemmli sample buffer, and immunoprecipitates resolved on 7.5% SDS-PAGE. Detection of Macrosialin mRNA-For Northern blotanalysis, RNA was prepared from RAW cells induced to differentiate with 25 ng ml" PMA for 24 h. Poly(A)' RNA was selected using magnetized oligo(dT) beads (Dynal). 4 mg of poly(A)' RNA or 25 mg of total, unselected RNA were loaded per lane, denatured in formaldehyde, electrophoresed in 1% agarose gels, transferred to Hybond-N' nylon (Amersham UK), uv-cross-linked in a Stratalinker (Stratagene), and hybridized with an antisense MS.l riboprobe generated from pBSMS. Recombinant Riboprobes-pBS-MS contained the first 390 bp of pMS.l in pBluescript. Antisense probes were generated from Sadlinearized pBS-MS by in vitro transcription with T 3 polymerase in the presence of ["PICTP. Polymerase Chain Reaction and Cloning-To determine the variations in cytoplasmic tail sequences of macrosialin, polymerase chain amplification reactions were performed on the following cDNA templates: first strandcDNA synthesized from the samePA' RNA source from thioglycollate-elicited peritoneal macrophages usedto construct the cDNA library, the macrophage library used for expression selection, and a cDNA library constructed from the RAW macrophage cell line. 100 ng of the cDNA sources were used as templates with 10 ng of the following primers: forward amplification primer a t 961 bp in macrosialinsequence 5'-TCCCTCTTGCTGCCTCTCATC-3';reverse amplification primer a t 1100 bp 5"GGTGGCTTACACAGTGGACTGG-3'. PCR conditions were as follows: 3 cycles of 92 "C for 2 min, 45 "C for 1 min, 72 "C for 1 min and 30 cycles of

92 "C for 1 min, 50 "C for 1 min, 72 "C for 1 min. PCR productswere treated with Klenow and polynucleotide kinase and blunt-end-cloned into phosphatase-treated EcoRV-cut pBluescript. Individual recombinant colonies were sequencedwithuniversalM13sequencing primers. DNA Sequencing-Double-stranded sequencing was conducted on the cDNA insert in pMS.l by dideoxy chain termination (Sanger et al., 1977) using sequence-specific oligonucleotides. Both strands of the cDNA insert were sequenced entirely. RESULTSANDDISCUSSION

To isolate a cDNA clone encoding murine macrosialin, a cDNA library was constructed from RNA prepared from thioglycollate-elicited peritoneal macrophages. The cDNA library was transiently expressed in COS cells, and cells expressing macrosialin cDNAs were isolated by panning with FA/11 mAb. Episomal DNA was recovered from the adherent cells, amplified in Escherichia coli, and reintroduced into COS cells. After one additional round of expression and selection, 12 of24 final round miniprep transfectants scored positive for staining with the FA/11 mAb. One type of cDNA insert was shown to have an insert of 1.2 kb, and one clone, pMS.l, was pursued in greater detail. mAb FA/11 immunoprecipitated a single protein of 85-110 kDa from "S-labeled proteins prepared from 5774 and RAW cells (Fig. 1).A slightly smaller protein of 75-100 kDa was immunoprecipitated from pMS.l-transfected COS cells (Fig. 1).The smaller molecular mass of macrosialin expressed in COS cells is probably due to inefficient or incomplete glycosylation of the expressed proteins. mAb FA/11 did not immunoprecipitate any protein from the B cell line NSO. Northern blotanalysis of poly(A)+-selected RNA from RAW cells showed a single species of approximately 1.3 kb (Fig. 2) that was slightly increased on PMA treatment. Macrosialin transcripts appear tobe rare, since a signal was only clearly seen in poly(A)-selected RNA and not in total RNA, where nonspecific binding of the pMS.l riboprobe to 28 and 18 S rRNA is evident (Fig. 2). The sequence of the cDNA insert in clone pMS.1 (Fig. 3) consists of 1243 bp ending in a poly(A) tract, preceded by a potential polyadenylation site at 1207 bp (Fig. 3, AATAAA, underlined). The predicted polypeptide sequence encoded by pMS.1, consists of 318 residues, and has thetypical featuresof a type I integral membrane protein. The sequence starts with an ATG at position 91 followed bya hydrophobic signal sequence

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FIG.1. Immunoprecipitation analysis of macrosialin. "SLabeled proteins from the stated cell lines were immunoprecipitated with mAb FA/11-coupled Sepharose. Lane I , molecular mass markers in kDa; lane 2, COS cells transfected with pMS.1 cDNA clone; lane 3, RAW (macrophage); lane 4, 5774 (macrophage); lane 5, NSO (B cell). Immunoprecipitates were resolved on 7.5% SDS-PAGE.

Macrosialin cDNA of 20 residues, which may be cleaved between glutamic acid 20 and aspartic acid 21 (von Heijne, 1986). The predicted mature form of macrosialin consists of an extracellular domain (271 residues), a hydrophobic transmembrane domain (25 residues), and a short cytoplasmic domain (2 residues). There are nine potential N-glycosylation sites (Am-X-Ser/ Thr). The predictedmass of the polypeptide backbone in clone pMS.1 is only 35 kDa, whereas the observed mass of themature glycoprotein is between 87 and 115 kDa. The extracellular domainhas numerous contiguousruns of serine, threonine, and proline, which could act as sites for attachment for 0-linked carbohydrate (Wilson et al., 1991). The largest contribution to the mass of the mature macrosialin comes from both 0- and N-linked carbohydrate; approximately40% from 0-linked and 20-25% from N-linked sugars, with only about one third coming from the polypeptide backbone (Rabinowitz and Gordon, 1991). Previous studies have defined the nature of the glycans borne by macrosialin on unstimulated and stimulated macrophages (Rabinowitz and Gordon, 1991). N-Linked sugars account for 21 kDa of the mass, and 0-linkedglycans account for 30-40% of the molecular mass of macrosialin. Both types 1

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FIG.2. Macrosialintranscript expression. Northern blot analysis of macrosialin expression in the murine macrophage line RAW. Lane I , 4 mg of poly(A+)RNA from untreated RAW cells; lane 2 , 4 mg of poly(A+) RNA from RAW cells treated with PMA (25 ng/ ml, 24 h); lane 3,25 mg of total RNAfrom untreated RAW cells; lane 4,25 mg of total RNA from RAW cells treated with PMA (25 ng/ml, 24 h). ..

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of glycans show extensive remodeling upon activation of macrophages, acquiring numerous terminal sialic acid residues and polylactosaminoglycans. A search of the NationalBiomedical Research Foundation protein data base (Wilbur and Lipman, 1983) disclosed homology to mouse lamp-1 and lamp-2 (Table I) (Chen et al., 1988; Cha et al., 1990) and othermembers of the lamp family (Fig. 4). Members of the lamp/lgp family are ubiquitously expressed glycoproteins located inlysosomal membranes (Fukuda, 1991a). The lamp proteins show a high degree of homology across different species; for example, mouse lamp-1 is more homologous to human lamp-1 than to mouse lamp-2 (Table I). Macrosialin shows more homology to lamp-1 proteins than lamp-2 proteins (TableI). However, macrosialin is most closely related to human CD68, showing 72.0% identity and 80.6% similarity (Table I and Fig. 4). The extracellulardomain of the lamp/lgp family has a bipartite organization, the two domains being divided by an extended proline hinge (Fukuda, 1991a) (Fig. 5). Macrosialin and CD68 share this bipartite structure, divided by the proline hinge; in both molecules, the area of greatest conservation is from the hinge to the cytoplasmic tail. The membrane-proximal domain is compact and probably globular; it contains four regularly spaced cysteines (36-37 residues apart), and,in the lamp family, intradomain disulfide bonds are formed between the first and second and between the 3rd and 4th cysteines (Carlssonand Fukuda, 1989; Arterburn et al.,1990). The equivalent domain of macrosialin also has 4 cysteines, all of which align with the equivalent cysteines in lamp-1 proteins (Fig. 4). The N-terminal domain of macrosialin is unrelated to the equivalent domain of the lamp/lgp family but is homologous to the corresponding domain in CD68; being very dense in serine and threonineresidues (43% Ser + Thr), characteristic of mucin-like molecules decoratedwith 0-linked glycan chains (Jentoft, 1990; Fukuda, 1991b). CD68 has a region of short repeat motifs in this domain, 12-18-12-18 (A-B-A'-B'). The corresponding region of macrosialin has a similar run of repeats, although they are shorter (6-15-8-17) and less internally conserved (designated by arrows in Fig. 3; indicated in detail in Fig. 6). The cytoplasmic tail in pMS.l is only 2 residues (RR*) and differs markedly from the tailsof CD68 and thelamp family, which are 10 residues andcontain a key tyrosine residue preceded by a smallside-group amino acid (alanine orglycine). This motif has been identified by in vitromutagenesis studies ......................... . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . .-. . . .

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FIG.3. Sequence analysis of macrosialin. Sequence of cDNA insert in clonepMS.1 is given. The N-terminal signal peptide, potential N-linked glycosylation sites(Am-X-Ser/Thr), and C-terminal transmembrane domain are underlined. Region of short peptide repeats is delineated with arrows (>>>>>)below the sequence.

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Macrosialin cDNA

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rophage RNA used to construct the cDNA library, and by PCR from the macrophage cDNA library and from a RAW cDNA library. PCR products were cloned and sequenced. Two types of cytoplasmic tail were seen; type 1,correspondCAA, . .), ing to the tail in pMS.1 (RR*;.. .CGC AGA was isolated from the original macrophage cDNA library and the RAW cDNA library; type 2, RRRQSTYQPL (. . .CGC CD68 Macrosialin mus-lamp1 ACA AGA CAA.. .), was isolated from the original macrohum-lamp-1 26.6 (44.4) 28.2 (51.5) 67.8 (77.5) phage library and from first strand cDNA. The type 2 tail is mus-lamp-1 25.2 (45.7) 27.6 (48.1) homologous the to 10-residue tail found in CD68 (55.0) hum-lamp-2 23.2 (42.9) 24.4 (46.7) 35.7 (RRRFSAYQAL) and the lamp/lgp family, and contains the mus-lamp-2 24.7 (46.9) 24.0 (46.7) 59.1 (36.0) key tyrosine residue. Four independent clones for the longer CD68 (70.2) 10-residue tail were isolated from the macrophage cDNA library used for panning, and two independent clones were 50 1 Cd6B ... isolated from first strand cDNA. Macrasialin ............................................. The short 2-residue tail isolated by transient expression Muslampl .EVLSFSASFL ........................................ Ratlgp120HRAPGhWlPLLLLLLAGLAHShPALFEVKDNNGTAC .... IUASFSASFL could have been an artifact introduced during cDNA synthesis Humlampl PIIIPRSRRRPL LLLLPVAVLR PHALSSAAW KVKNGNGTAC IHANFSAAFS or later duringthe two rounds of expression selection in COS 51 100 .WLAVLfSG Cd68 ALLGLLAAW . . . TGNDCPHKKS . . . . . . ATLLPSFTVT . cells. However, as the same RR* tail (resulting from a single Macrosialin . . . . . . . . . . .MRLPVCL.. ILLGPLIAQG TEEDCPHKKA VTLLPSFTMT base change; AGA to TGA) was re-isolated from a different nullampl TTYETRNGSQ IVNISLPASA EVLKNGSSCG KENVSDPSLT ITFGRGYLLT RatlqplZO TTYDAGHVSK VSNMTLPASA EVLKNSSSCG EKNASEPTLA ITFGEGYLLK cDNA library, it seems possible that this tail variant does Humlampl VNYDTKSGPK NMTFDLPSDA TWLNRSSCG KENTSDPSLV IAFGRGHTLT indeed exist. 150 101 Cd68 PTVTESTGTT SHRTTRSHKT TTHRTTTTGT TSHGPTTATH NPTTTSHGNV Macrosialin may therefore exist in two forms: one solely M a c r o s i a l i n PTATEST . . . . . . . . . . . . . . . . . . . . . . . . . . ASPTTSH RPTTTSHGNV localized to the plasma membrane (RR*) and the other tarnuslampl LNFTKNTTRY SVQHMYfTYN LSDTEHFPNA ISKEIYTM.. DSTTDIKADI Ratlgp120 LTfTKNTTRY SVQHMYfTYN LSDTQFFPNA SSKGPDTV.. DSTTDIKADI geted tothe lysosomes (RRRQSTYQPL).Thereare two Humlampl LNFTRNATRY SVQLHSPVXN LSDTHLFPNA SSKEIKTV.. ESITDIRADI possible waysin which this could arise. First, macrosialin may 151 200 Cd68 TVHPTSNSTA TSQGPSTAT. HSPATTSHGN ATVHPTSNST ATSPGFTSSA be encoded by two closelyrelated genes with slightly different H a c r o s i a l i n TVHTSSGPTT VTHNPATTTS HGNATISH.. RTVSPTTNGT A T S P K S S T V nwlampl NKAYRCVSDI RVYMKNVTW LRDATIQRYL SSGNFSKEET HCTQDGPSPT cytoplasmic tails; second, there maybe a single gene with Ratlqp120 NKTYRCVSDI RVYUKNVTIV LWDATIQAYL PSSNTSKEET RCPQDQPSPT separate exons encoding the two tail variants anddifferential Humlampl DKKYRCVSGT Q V H W T V T LHDATIQAYL SNSSFSRGET RCEQDRPSPT splicing gives rise to the two variants. A precedent exists for 250 the first of these in the low affinity receptor for IgG, FcRIII, or CD16, which exists in two isoforms differing by only 1 nucleotide in the cytoplasmic domain (Ravetch and Perussia, 1989). The two isoforms are encoded by two nearly identical 251 3ou but separate genes that lie very close together in the genome. These genes are transcribed in a cell-type-specific manner and generate either a phosphatidylinositol-anchored receptor in neutrophils or a transmembrane-anchoredreceptor in nat301 .SNSSIIL Cd68 W L S Y M A V E Y NVSFPHAAKW TF'SAQNASLR DLQAPLGQSF ural killer cells, lymphocytes, and macrophages. For macro.GNASIVL Hacrosialin VYLDYMAVEY NVSFPQAAQW TfElRQNSSLR ELQAPLGQSF TEEHIFI' sialin, it will be interesting to determine the relative expresMvslampl FFLO..GVRL NMTLPDALW TfSISNHSLK ALQRTVGNSY Ratlgp120 FFLQ..GVQLNMTLPDAIEPTFSTSNYSLKALQASVGNSYSEEHlFV sion of these two tailvariants in resting uersus activated Humldmpl PFLQ..GIQLNTILPDARDP A F W G S L R ALQRTVGNSYAEEHVPV j50 macrophages and whether this influences the amount of intra351 4011 PSD.RS1LLP LIIGLILLGL Cd68 SPAVHLDLLS LRLQAAQLPH TGVFGQSF uersus extracellularly expressed glycoprotein. NRD.QSLLLP LIIGLVLLGL naciosialin SPAVHLDLLS LRLQAAQLPD KGHPGPCF VQDGNNMLIP IAVGGALAGL On the basis of the sequence and domain homologies prenuslampl SXIILSLNVFS VQVQA5XV.D SDRFGSVE RatlgpIlO SKALALNVFS VQVQAFRV.E SDRFGSVE VQDGNNMLIP IAVGGALAGL sented here, macrosialin and its humanhomologue CD68are LLDENSTLIP IAVGGALAGL numlampl TKAFSVNIFK VWV0AFKV.E GGQFGSVE new members of the lamp/lgp family of lysosomal associated glycoproteins. However, immunohistochemical studies reveal a difference between the expression profiles of macrosialin and CD68; FA/11 staining has only been detected in macrophages, Langerhans cells, and isolated dendritic cells (Gordon FIG. 4. Alignments of macrosialin with CD68 and the lamp et al., 1992; Rabinowitz and Gordon, 1991), whereas CD68 is family. A multiple alignments with representative members of the lamp-1 family are shown. Alignments were produced using the GAP expressed at very low levels in most cell types but is abundant program based on the algorithm of Needleman and Wunsch (Univer- in macrophages and many tumor cell lines (Pulford et al., sity of Wisconsin Genetics Computer Group package; Devereux et al. 1990). One explanation could be that the CD68mAbs are (1984)). The proline hinge is boxed, and the transmembrane regions detecting epitopes in other related proteins or lamps, whereas are underlined. Asterisks and/or boxes indicate the aligned cysteines the FA/11 epitope is absolutely specific for macrosialin. In that have structural significance for lamp-1. (I) is put between addition, the CD68 studies were carried out with the more identical amino acids. (:) is put between similar amino acids whose comparison value is greater to or equal to 0.5. (.) is put between sensitive APAAP technique, whereas the macrosialin studies similar amino acids whose comparison value is greater than or equal to date have utilized avidin-biotin-peroxidase. Alternatively, to 0.10. the expression profiles could indeed reflect a real difference between macrosialin and CD68; macrosialin may have a role to play a dominant role in lysosomal targeting (Williams and only in macrophages, whereas CD68 may play a role in enFukuda, 1990; Eberle et al., 1991; Harter and Mellman, 1992). docytosis or lysosomal traffic that is an essential, "houseThe lack of homology in this region prompted us to look keeping" activity in all cells, and this activity has been exfor variant cytoplasmic tail sequences by PCR amplification panded to become a dominantpart of the specialized function from first strand cDNA constructed from the original mac- of phagocytic cells. Alignment of CD68,macrosialin, and mus-lampl with lamp proteins Alignments were produced using the GAP program based on the algorithm of Needleman and Wunsch (University of Wisconsin Genetics Computer Group package; Devereux et al. (1984)). The percentage amino acid identities and the percentage similarities are tabulated, the percentage similarities being recorded in parentheses.

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FIG. 5. Model for the domain organization of the lamp, macrosialin, and CD68 glycoproteins.

+ +

rodlike structure of leukosialin, CD43, could protrude above the glycocalyx of the cell and allow multiple glycan chains to TnrhlaSerProTnrThr SerH1SATgPTOlnrlnrTnrLySHISGly*snValT-~~~~l~~~ be accessiblefor binding (Cyster et al., 1991). It has been directly demonstrated that lamp-1 on leukemic cells carries B' A ' sialylated fucosylated polylactosaminoglycans that can bind h to E-selectin (Saitoh et al., 1992). In addition, a 110-120-kDa 'lo 40 TnrSe'Se'GlyPrOThTThrVai TOriilSA~'iPr~AlilThTThTTPlrSerHlgGiyASnAl~Tn~~I~ie~ glycoprotein on myeloid cells with similar properties to maFIG.6. Organization of the amino acids in the A-B-A'-B' crosialin/CD68 hasrecently been identified as a potential repeat motif. The numbers nboue the amino acids indicate their presenter of glycan-ligands to P-selectin (CD62) on vascular position in the mature macrosialin protein. endothelium(Moore et al., 1992). This glycoprotein is not lamp-1, lamp-2, or CD43, and we are currently investigating The intracellular role of the lamp family is unknown. It is whether it could be macrosialin or CD68. proposed that the extensive 0-glycosylationof these proteins Macrosialin and CD68 are the first examples of the lamp protects the core protein and other closely associated mem- family with a restricted cell-type-specific expression pattern. brane proteins from enzyme attack in the lumenof the lyso- To date, all existing members of the family are constitutively some (Jentoft, 1990; Rabinowitz and Gordon, 1992). The more and ubiquitouslyexpressedinall cell types.Macrosialin/ restricted expression of macrosialin/CD68 suggests its inCD68 have clearly diverged from the main members of the volvementinspecialized mononuclear phagocyte functions family by replacingtheN-terminaldomain witha novel such asphagocytosis of pathogens and antigen processing and mucin-like domain. This may shed some light on the relative presentation. The overexpression of a mucin-protected lamp functions of the two distinct domains. Thecommon globular (macrosialin/CD68) could be essential for the survival of the membrane-proximal domain could play some general role in lamp and the survival of other associated glycoproteins, in lysosomal metabolism, whereas the membrane-distal domain the harsh hydrolytic environment of the lysosomes. function associatedwith the As CD68 and macrosialin also appear on the cell surface, may have amorespecialized phagocytic activities of macrophages such as bindingforeign they may also have extracellular functions. One possible extracellular role forthese molecules could residein their exten- organisms (yeasts,viruses, bacteria, and protozoan parasites) sive glycan decoration. Recently, it has been proposed that and killing tumor cells. The expression of both macrosialin one of the functions of heavily glycosylated hematopoietic and CD68 in dendritic and Langerhans cells may point to a membrane proteins such as CD43/leukosialin (Killeen et al., role for these molecules in antigen processing, which is a key 1987; Shelley et al., 1989; Cyster et al., 1990), CD34 (stem cell function of these specialized cells. Domain swaps between the antigen) (Sutherland et al., 1988; Simmons et al., 1992), and lamps andmacrosialin/CD68 can now be carried out toinvesSgp50 or GlyCAM-1 (the high endothelial ligand for L-selec- tigate these ideas. tin) (Lasky et al., 1992) is to present carbohydrate ligands to Acknowledgment-We thank Dr. PaulCrocker for discussions and P- critical selectins(Williams, 1991). Theselectins(E-,L-,and review of the manuscript. selectin) area family of membrane glycoproteins that mediate REFERENCES cell-cell adhesion events, especially those involving the attachment of circulating cellswithvascular endothelium Arterburn, L. M., Earles, B. J., and August, J. T.(1990) J. Eiol. Chem. 265, 7419-7423 (Stoolman, 1989). Theyshare a commonoverall structure Brandley, B. K., Swiedler, S. J., and Robbins, P. W. (1990) Cell 63,861-863 having an N-terminal C-type lectin domain that contains theCarlsson, S. R., and Fukuda, M. (1989) J. E d . Chem. 264,20526-20531 binding site(s) forspecific carbohydrate ligands. E-selectin Cha, Y . , Holland, S. M., and August, J. T.(1990) J. E d . Chem. 265,50085013 and P-selectinexpressed on endothelialcells, bind a structure Chen, J. W., Cha, Y . , Yuksel, K. U., Gracy, R. W., and August, J. T . (1988) J . E d . Chem. 263. 8754-8758 ,~ . .~ resembling sialylated Lewis X, presented to themby circulatJ. C., Somoza, N., Killeen, N., andWilliams, A. F. (1990) Eur. J. ing leukocytes (Bradley et al., 1990). The molecules bearing Crster, Immunol. 20,875-881 Cyster, J. C., Shotton, D. M., and Williams, A. F. (1991) EMEO J. 10,893these glycan ligandshave not yetbeenidentified, but the 902 heavilyglycosylatedglycoproteins listed above havebeen Devereux, J., Haeberli, P, and Smithies, 0. (1984) Nucleic Acids Res. 12,387proposed as potential candidates. For example, the extended 396 A

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Macrosialin cDNA

9666

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