Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Cytokinesis wikipedia , lookup
Cell membrane wikipedia , lookup
Cell culture wikipedia , lookup
Cellular differentiation wikipedia , lookup
Extracellular matrix wikipedia , lookup
Tissue engineering wikipedia , lookup
Cell encapsulation wikipedia , lookup
Signal transduction wikipedia , lookup
Endomembrane system wikipedia , lookup
From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Molecular Cloning of CD68, a Human Macrophage Marker Related to Lysosomal Glycoproteins By Claire L. Holness and David L. Simmons CD68 is a 1 IO-Kd transmembrane glycoproteinof unknown function highly expressed by human monocytes and tissue macrophages. We have isolated cDNA clones encoding CD68 from a U937 cDNA library by transient expression in COS cells and panning with the anti-CD68 monoclonal antibodies (MoAbs) Y2/131, Y1/82A, EBMI 1, and Ki-M6. CD68 transcripts are constitutively present in the promonocyte cell line U937 and are upregulated by phorbol myristic acid (PMA). By contrast, CD68 transcripts are absent or present at very low levels in many hematopoietic lines including KG1, CEM, and K562, but can be induced by exposure to PMA. The cDNA sequence predicts a type I integral membrane protein of 3 5 4 residues with a heavily glycosylated extracellular domain of 2 9 8 residues contain- ing nine potential N-linked glycosylationsites and numerous potential 0-linked glycosylation sites. The extracellular domain consists of two distinct regions separated by an extended proline hinge: a membrane-distalmucin-like domain containing short peptide repeats and consisting of 54% serine and threonine residues; and a membrane proximal domain that has significant sequence homology to a family of lysosomal/plasma membrane shuttling proteins known as the lamp 1 group. CD68 is a member of a growing family of hematopoietic mucin-like molecules, including leukosialin/CD43, the stem cell antigen CD34, and the lymph node high endothelial ligand for L-selectin GlyCAM-1. 0 1993 by The American Society of Hematology. T family of hematopoietic mucin-like molecules, including leuko~ialin~~~~~/CD43,*' CD34," and the lymph node high endothelial ligand for L-selectin GlyCAM- I .23 ISSUE MACROPHAGES are derived from cells of the mononuclear phagocytic system, and are thought to represent the end-stage of differentiation of circulating monocytes.' Macrophages are widely distributed throughout the body and display great structural and functional heterogeneity, reflectingconditions within their local environment. Tissue macrophages are involved in many immune functions, the most significantbeing phagocytosis of foreign and necrotic material, and antigen processing and presentation.2 The surface phenotype of mononuclear phagocytes is poorly defined by specific monoclonal antibodies (MoAbs), compared with other hematopoietic lineages. The majority of antigens defining the mononuclear phagocyte lineage are in fact absent from tissue macrophage^.^ However, antibodies to CD68 recognize a 110-Kd glycoprotein on tissue macrophages and blood monocyte^.^‘^ A wide range of tissue macrophages are recognized, including germinal-center macrophages, alveolar macrophages, macrophages from human tonsils, splenic red pulp, connective tissue of the dermis, Kupffer cells of liver, and blood To better understand the structure and expression of this important macrophage antigen, we have isolated cDNA clones encoding CD68 by transient expression in COS cells and immunoselection" using anti-CD68 MoAbs. Two types of cDNA clone were isolated, differing by the insertion of two short segments in the N-terminal portion of the extracellular domain. CD68 has been localized to endosome- or lysosome-like structures by immunoelectron microscopy.I I Consistent with this, the sequence of CD68 shows homologies to a family of lysosomal/plasma membrane shuttling proteins, typified by the lamp 1 group (human lamp-1,I2 mouse lampl,I3 chicken lep100,14-'6and rat lgpl2O"). These proteins are major components of lysosomal membranes and shuttle in vesicles between lysosomes, endosomes, and the plasma membrane.16 While the intracellular function of the lamps is unknown, an extracellular role as presenters of carbohydrate ligands to selectins has recently been reported.I8 Human lamp- 1 and lamp-2 are decorated with sialylated fucosylated polylactosaminoglycans that are ligands for E-selectin and have been implicated in tumor cell metastasis'*; lamps are over-expressed on the surface of metastatic compared with nonmetastatic cell lines. CD68 is a member of a growing Blood, Vol 81, No 6 (March 15), 1993: pp 1607-1613 MATERIALS AND METHODS Cell lines and culture conditions. The cell lines U937, KG 1, K562, and CEM were obtained from the Imperial Cancer Research Fund (ICRF)cell bank. All cells were grown in Dulbecco's modified Eagle's medium (DMEM)/lO%fetal calf serum. U937 cells were maintained at a density of 5 X IO5 mL-' and induced as follows: 25 ng mL-' PMA for 24 hours, or 100 U mL-' y-interferon for 24 hours. Library construction and screening. A cDNA library was constructed in the expression vector 7rH3M" from RNA prepared from induced promonocytic U937 cells (10 nmol/L phorbol myristic acid [PMA] for 24 hours). Cells expressing CD68 were isolated with the anti-CD68 MoAbs Y2/131, Y1/82A, EBMIl, and Ki-M64,5(CD68 MoAbs were a gift from D. Mason, John Radcliffe Hospital, Oxford, UK) and panned on goat anti-mouse dishes." Episomal DNA was recovered from the panned cells and the expression-panning cycle was repeated two times to obtain cDNA clones designated CD68.1 and CD68.2. Expression of CD68 on the surface of transfected COS cells was detected by indirect immunofluorescence using MoAbs Y I / 82A and Y2/ I3 1 and fluorescein-conjugated goat anti-mouse IgG antibody (Sigma, St Louis, MO). Suvface labeling and immunoprecipitations. Cells were surface labeled with '*'[I] using glucose oxidase lactoperoxidase as Enzymobeads (BioRad, Richmond, CA) according to the manufacturer's protocols, extracted with a 1% NP40 buffer (1 50 mmol/L NaCl, 1.O% NP40, 50 mmol/L Tris pH 8.0 and phenylmethylsulfonyl fluoride [PMSF] 50 pg/mL) and immunoprecipitationswere performed using From the Cell Adhesion Laboratory, Imperial Cancer Research Fund, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, UK. Submitted August 6, 1992; accepted November 9, 1992. Supported by the Imperial Cancer Research Fund (UK). Address reprint requests to David L. Simmons, PhD, ICRF Labs, Institute of Molecular Medicine, John Radclffe Hospital, Headington, Oxford, OX3 9DU UK. The publication costs of this article were deji-ayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.section 1734 solely to indicate this fact. 0 1993 by The American Society of Hematology. 0006-4971/93/8106-0012$3.00/0 1607 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. HOLNESS AND SIMMONS 1608 MoAbs Y2/131, EBMII, KP1, and control MoAb anti-VCAM-1 4B2 (British Bio-technology,Oxford, UK), and affinity-isolated goat anti-mouse agarose (Sigma). The immune complexes were centrifuged through a cushion of 30% sucrose in 1% NP40 buffer and then washed three times in the 1% NP40 buffer (above). The denatured proteins were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Recombinant riboprobes. pGAPDH, a gift from Sean McCarthy (Molecular Oncology ICRF, IMM, Oxford, UK), contained 120 bp of GAPDH cDNA in pBluescript. Antisense probes were generated from HindIII linearized pGAPDH by in vitro transcription with T3 polymerase in the presence of 32[P]-CTP.pBSCD68 contained the first 350 bp of pCD68.1 in pBluescript. Antisense probes were generated from HindIII linearized pBSCD68 by in vitro transcription with T7 polymerase in the presence of ”[PI-CTP. pBSICAM 1 contained 235 bp of ICAM1 cDNA (Sma I at 685 to Pst I at 920) in pBluescript. Antisense probes were generated from SmaI linearized pGAPDH by in vitro transcription with T7 polymerase in the presence of ”[PI-CTP. Detection of CD68 mRNA. For Northern blot analysis, RNA was prepared from U937 cells, polyA+ RNA selected using magnetized oligo-dT beads (Dynal, Oslo, Norway) and 4 pg polyA+RNA loaded per lane, denatured in formaldehyde, electrophoresed in 1% agarose gels, transferred to Hybond-N+ nylon (Amersham, Amersham, UK), UV cross-linked in a Stratalinker (Stratagene, La Jolla, CA), and hybridized with an antisense CD68. I riboprobe generated from pBSCD68. For RNase protection assays, 25 pg total RNA was hybridized overnight in 400 mmol/L NaCl and 80%formamide at 45°C with I X IOs cpm (5 ng) of riboprobe and then digested with 40 pg/ mL RNaseTI at 25°C. Protected fragments were resolved on 6% polyacrylamide/S mol/L urea gels and detected by autoradiography. DNA sequencing. Double-stranded sequencing was conducted on the CD68.1 and CD68.2 by dideoxy chain termination24using sequence-specific oligonucleotides. Both strands of each cDNA insert were sequenced entirely. RESULTS AND DISCUSSION To isolate a cDNA clone encoding the tissue macrophage marker defined by the CD68 MoAbs, a cDNA library was constructed from RNA prepared from U937 cells induced to differentiate with PMA. The cDNA library was transiently expressed in COS cells and cells expressing CD68 cDNAs were isolated by panning with four of the anti-CD68 MoAbs from the Fourth Leukocyte Typing Workshop4.’ (Y2/ 131, EBMl1, Ki-M6, and Y 1/82A). Episomal DNA was recovered from the adherent cells, amplified in Escherichia coli, and reintroduced into COS cells. After three rounds of expression and selection, 14 of 16 final round miniprep transfectants scored positive for staining with the CD68 MoAb pool. Two types of cDNA insert were seen among these clones: CD68.1 (6 of the 14 positives) had an insert of approximately 1.5 kb, CD68.2 (8 ofthe 14 positives) had an insert of approximately 1.0 kb. Both cDNA clones transiently expressed in COS cells gave positive surface immunofluorescence with MoAbs Y 1/82A, Y2/ 13I , and EBM 1 l 9 (data not shown). Neither clone gave positive surface staining with MoAb K P l , which probably recognizes a carbohydrate-based epitope.’ Two MoAbs (EBM I 1 and Y2/ 131) were selected for immunoprecipitation analysis as these were known to be efficient MoAbs from previous work.’ ‘*’[I]-labeled membranes from CD68-transfected COS cells were immunoprecipitated with MoAbs EBM 1 I and Y2/ I3 I , and two different sized proteins were observed depending on the cDNA clone used (Fig I); pCD68.1 encodes a protein of approximately 1 I O Kd, while CD68.2 encodes a protein of approximately 80 Kd. KPI failed to immunoprecipitate any protein from the transfectants, consistent with the fact that it may recognize macrophagerestricted glycan-based epitopes. Northern blot analysis of polyA’ selected RNA from U937 cells showed a single species of approximately 2.0 kb (Fig 2A); that was increased by PMA treatment. Transcripts of equivalent size were also detected in RNA from HL60 cells and expression was augmented by treatment with PMA (data not shown). RNAase protection assay confirmed the presence of CD68 transcripts in uninduced U937 cells (Fig. 2B). yInterferon (y-IFN) did not significantly upregulate CD68 expression whereas the expression of ICAM- 1 was induced (Fig 2B). CD68 transcripts were absent from resting KG1 cells (myeloblastic leukemia) and CEM (T lymphoblastic leukemia) and present at low levels in K562 cells (erythroleukemia) (Fig 2C). CD68 transcripts were induced in all these lines by PMA treatment. Thus, CD68 is constitutively expressed in promonocyte cells but can be expressed in most hematopoietic cell lines by phorbol-induced differentiation. The sequence of the cDNA insert in clone pCD68.1 (Fig 3) consists of 1,719 bp. The cDNA insert in pCD68.2 is 972 bp. Both clones have identical 5’ untranslated sequences but differ at their 3‘ ends; the CD68.1 insert ends in a polyA tract and has Alu I repeats in the 3’ untranslated sequence; the CD68.2 insert has no such sequences and was probably truncated in vitro during cDNA synthesis or as a result of transient expression and selection in COS cells. The predicted polypeptide sequence encoded by pCD68.1 consists of 354 residues and has the typical features of a type I integral membrane protein. The sequence starts with an ATG at position 13 followed by a hydrophobic signal sequence of 20 residues, which may be cleaved between glycine21 and asparagine-22.25 The predicted mature form of CD68. I consists of an extracellular domain (298 residues), a hydrophobic transmembrane domain (25 residues), and a short cytoplasmic domain ( I O residues). Clone CD68. I has two separate apparent “insertions” relative to clone CD68.2, in the N-terminal region of the extracellular domain (Fig 4A). The inserted sequences do not break the reading frame with the result that CD68.1 has an open reading frame of 354 residues, while CD68.2 has an open reading frame of only 297 residues. The inserted segments in CD68.1 occur at positions 59 through 139 (27 residues) and 256 through 346 (30 residues). Neither sites are flanked by the consensus sequences of eukaryotic exon/intron boundaries so the insertions are unlikely to represent unspliced introns. In addition, both sequences do not interrupt the open reading frame. Interestingly, the second inserted sequence occurs in a region of tandem repeats in the protein sequence. In CD68.1 the pattern is: 12-18-12-18 (A-B-A‘-B’), while in CD68.2 the pattern is 12-18 (A-B), so that the central part of the repeat ( 18- 12, B-A‘) is missing (Fig 4B). As this region consists of direct, not inverted repeats, it is unlikely to have arisen through the formation of secondary structures From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 1609 MOLECULAR CLONING OF CD68 Mr kd 1 2 3 4 5 6 7 8 20092 - - 67 Fig 1. Immunoprecipitation analysis of CD68. ‘Z6[lllabeled membrane extracts from COS cells transfected with the stated cDNA clones were immunoprecipitated with MoAbs: lanes 1 through 4, pCD68.1; lane 1, control MoAb BB-VCAM-1, 482; lane 2, MoAb KP1; lane 3, MoAb EBMl 1; lane 4, MoAb Y2/131; lane5, pCD68.2. MoAb Y2/131; lane 6, pCD68.1, MoAb EBMl 1;lane 7, pCD68.2, MoAb KP1; lane 8, pCD68.2, control MoAb BB-VCAM-1, 402. - 43 30 20 (stem/loop) during cDNA synthesis. In addition, the repeats are not identical (A =/= A’, B =/= B). Thus, we are left with the intriguing possibility that differential splicing of “mini-exons” yields at least two differ- B .-., ,I.,2- 3 4-= ~ I. -CD68 ent CD68 proteins. As only the larger 1 IO-Kd protein was observed from human spleen h~mogenates,~ the 80-Kd pCD68.2 encoded product may be the minor spliced transcript and hence minor protein species. A detailed study of C 1 2 3 4 5 6 7 - --.-CD68 28S-CAM 1 18s- ri -GAPDH Fig 2. CD68 transcript expression. (A) Northern blot analysis of CD68 expression in the promonocyticleukemic line U937. Four micrograms of poly A+ RNA from: lane 1, uninduced U937 cells; lane 2,4 pg of poly A+ RNA from U937 cells treated with PMA (25 ng/mL, 2 4 hours). (B) Ribonuclease protection analysis for CD68 mRNA and ICAM-1 mRNA in 25 pg of total RNA from U937 cells. As a loading control, GAPDH mRNA was detected in the same RNA samples: lane 1, U937 cells probed for CD68 mRNA; lane 2, U937 treated with y-IFN (100 U/mL. 12 hours) and probed for CD68 mRNA; lane 3, U937 cells probed for ICAM-1 mRNA; lane 4, U937 treated with y-IFN (100 U/mL, Ribonuclease protection analysis for CD68 mRNA and, as a loading control, GAPDH mRNA. 12 hours) and probed for ICAM-1 mRNA. (C) Assays were performed on 25 pg of total RNA from: lane 1, CEM (human T-cell leukemia) treated with PMA; lane 2, untreated CEM; lane 3. KG1 (myeloblastic leukemia/acute myeloid leukemia) cells treated with PMA; lane 4, untreated KG1 cells; lane 5, K562 (human erythroleukemia)treated with PMA; lane 6, untreated K562; lane 7, untreated U937. From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 1610 HOLNESS AND SIMMONS 1 1 GCGGGCGGTTCAGCCATGAGGCTGGCTGTGCTTTTCTC~GGCCCTGCTGGGGCTACTGGCAGCCCAG~ACA~TGACTGTCCTCA~TCAGCTACTTTGCTGCCATCC 121 36 TTCACGGTGACACCCACGGTTACAGAGCACTGGAACTGGAACAACCA~CACAGGACTACCAAGAGCCACAAAACCACCACTCACA~~CCACCACAGGCACCACCAGCCAC~CCCACG PheThrValThrProThrValThrGluSerPhrGl~h~hrSerHisAr~hr^ThrLysSerHisLysTh~~hrHis~~~h~h~hrGl~hrThrSerHisGlyProThr MetArgLeuAlaValLeuPheSerGlyAlaLeuLeuGlyLeuLe~l~laGlnGl~hrGlyAsnAs~sProHisLysLysSerAlaT~Leu~uProSer ____________________________________ 241 76 >>>>>>>>>>>>>>>>>> ACTGCCACTCACAACCCCACCACCACCAGCCATGGAAACGTCACAGTTCATCCAAC~GC~TAGCACTGCCAC~GCCA~CCCTCAACTGCCACTCACAGTCCT~CACCACTAGT ThrAlaThrHisAsnProT~~~SerHisGlyAsnValThrValHisProThrSerAsnSerThrAlaThrSerGlnGlyProSerT~AlaThrHisSerProAlaTh~hrSer >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>---CHO--->>>>>>>>>>>>>>>---CHO--->>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> 361 116 CATGGAAATGCCACGGTTCATCCAACAAGCAACAGCACTGCCACCAGCCCAGGATTCACCAGTTCTGCCCACCCAGAACCAC~CCACCC~TCCGAGTCCTAGCC~CCTCCAA~G 481 156 ACCATTGGAGACTACACGTGGACCAATGGTTCCCAGCCCTGTGTCCACCTCCAAGCCCAGATTCAGATTCGAGT~TGTACACAACCCA~T~~GAGGCC~ATCTCTGTA 601 196 721 236 841 276 961 316 HisGlyASnAlaThrValHisProThrSerAsnSeirhrAlaThrSerProGlyPheThrSerSerAlaHisPrffiluProProProProSerProSerProSerProThrSerLysGlu >>>>>>---CHO--->>>>>>>>>>>>>>>---CHO--->>> ThrIleGlyAsp~r^ThrTrpT~AsnGlySerGlnProCysValHisLeuGlnAlaGlnIleGlnIleArgValMetTy~hirhrGlnGlyGlyGlyGluAlaT~lyIleSerVal ---CHO--CTGAACCCCAACAAAACCAAGGTCCA~GC~TGA~TGCCCATCCCCACCTGCTTCTCTCATTCCCCTATGGACACCTCAGCTTT~TTCATGCA~CCTCCAGCAGAAGGTT LeuAsnProAsnLysThrLysValGlnGlySerCysGluGlyAlaHisProHisLeuLeuLeuSerPhePro~~lyHisLeuSerPheGlyPheMetGlnAspLeuGlnGlnLysVal ---CHO--GTCTACCTGAGCTACATGGCGGTGGAGTACAATGTGTCC~CCCCCACGCAGCAAAGTGGACATTCTCGGCTCA~TGCATCCCTTC~~TCTC~GCACCCCT~AGAGCTTC ValTyrLeuSe~rMetAlaValGlu~rAsnValSerPheProHisAl~laLysTrpThrPheSerAlaGlnAsnAlaSerLe~gAspLeuGl~laProLeuGlyGlnSerPhe __ - - -CHO.._-CHO--AGTTGCAGCAACTCGAGCATCATTC~TCACCAGCTGTCCACCTC~CCTGCTCTCCCTGA~TCCAGGCATGACGGC~CTCAGCT~CCCACACA~TC~~AAAGTT~CTCCTGCCCC SerCysSerAsnSerSerIleILeLeuSerProAlaValHisLeuAspLeuLeuSerLeuArgLeuGlnAl~laGlnLeuProHisThrGlyValPheGlyGlnSerPheSer~sPro ---CHO--AGTGACCGGTCCA~TTGCTGCCTCTCTCATCATC~CTGATCC~CTTGGCCTCCTCGCCCTGGTGCTTATTGCTTTC~~ATCC~~C~CCATCCGCCTACCAGGCCCTCTGA SerAspArgSerIleLeuLeuProLeuIleIleGlyLeuIleLeuLeuGlyLeuLeuAlaLeuValLeuILeAlaPheCysIleIleArgArgArgProSerAlaTy~l~laLeu*** 1081 1201 1321 1441 1561 ___-----_______---___~____-------_ __________________________________ T M . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GCAmGCTTCAAACCCCAGGGCACTGAGGGGGTTTGGGGTGTGGT~~TACCCTTATTTCCTCGACACGCCGCTGGC~AAAGACAATGTTATTTTCC~CCCTTTCTT~GAA CAAAAAGAAAGCCGGGCATGACGGCTCATGCCTGT~TCCCA~ACTTT~GGCTGAG~AGGT~ATCACT~~~AGG~~GAGGCCA~CCTAGCCAACAT~~TAAACA CTGTCTCTACTAAAAATACACAGGTGTGGCGGCGT~TCCCATGCTAACCTGTAATCCCAGCTACTTG~GGCTGA~AGAGC~TTGAACCC~GT~~TTGCAG TGAGCCTGTCATCGCTCCACTGAGCCAAGATCGCTCCCACTGCACTCCAGCCTGGGCGACAGAGCCAGACTGTCTCAAATAAATAAATATGAGATAA~AG~~GAA~A~G AGAATTTTATTAAATGTGACGAACTGCCCCCCCCCCCCCCCCCA~AGGAGAGCA~T~ATGTAAATCTTTGAC~~TTTCCTTGC~CT~CA~~~GTCCATGAGT - -- - - -- 1681 TTCTTGCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Fig 3. Sequence analysis of CD68. Sequence of cDNA insert in clone CD68.1. N-terminal signal peptide, potential N-linked glycosylation sites (Asn-X-Ser/Thr), and C-terminal transmembrane domain (TM), are underlined. The "direct repeat sequence region" in CD68.1 (half of which is deleted in CD68.2) is delineated with arrows (>>>>>) below the sequence. a>>>>>>>-c->>>>>-CI>>>>>>>>>>>-, A M R L A V L F S G A L L G L L A A Q G T G N D C P H K K S A T L L P S F T V T ~ T S Q G P S T A T H S P A T T S H G N A T V H F * ~ S N S T A ESTGTTSHRTTKSHKTTTHRTTTTGTTSHGPTTSHGPTTATmPT MRLAVLFSGALLGLLA TTSHGNATVHFTSNSTA 1 16 first insertion 42 82 second insertion 113 B - 12 A TSHGPTTATHNP A' TSQGPSTATHSP 18 + TTTSHGNVTVHPTSNSTA ATTSHGNATVHPTSNSTA B B' Fig 4. Differences between CD68 proteins encoded by pCD68.1 and pCD68.2. (A) Deduced protein sequences in N-terminal region of pCD68.1 and pCD68.2. (B) Representationof the direct repeat sequences differing between clones CD68.1 and CD68.2. From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 1611 MOLECULAR CLONING OF CD68 CD68 LAMP 1 CD68 LAMP1 CD60 LAMP1 CD60 LAMP 1 CD60 LAMP 1 CD60 LAMP 1 CD60 Fig 5. Alignment of CD68 polypeptide sequence with human lampl CD68 and lampl protein sequences were aligned using the GAP program based on the algorithm of Needleman and Wunsch (University of Wisconsin Genetics Computing Group package3’). The proline hinge is boxed and the transmembrane regions are underlined; (*) indicates the aligned cysteines that have structural significance for lamp12s~30; (I) is put between identical amino acids; (:) is put between similar amino acids whose comparison value is >0.5; (.) is put between similar amino acids whose comparison value is 20.1 0. . 1 CD68 ’ CD68 LAMP 1 .................. MRLAVLFSGALXLLAAQGT ............ 20 I . I I : I : : : .:.I1 MAPRSARFPLLLLLPVAAARPHALSSAAMFMVMGNGTACIMANFSAAFS 50 ...GNDCPHKKSATLLPS .....FTVTPTVTESTGTTSHRT..TKSHKTT :..:..I. . Ill :. ..... I . I : . . I . .::I. I 60 VNYDTKSGPKNMTFDLPSDATWLNRSSCGKENTSDPSLVIAFGRGHTLT 100 THRTTTTGTTSHGPTTATHN : I ... I . . . . I ....... PTTTSHGNVTVHPTSNSTATSQ. i. .I.: I1 .... : I . : 102 LNFTRNATRYSVQLMSFVYNLSDTHLFPNASSKEIKTVESIDK 150 . . . .GPSTATH.SPATTSHGNATVHPTSNSTATSPGFT ....... SSAHP ..:I..I . . I . . : I [ : : : .... 1 . 1 I I.. : 140 KYRCVSGTQVHMNNVTVTLHDATIQAYLSNSSFSRGETRCEQDRPSPTTA 200 t PPPPSPSPSPT .I:IIIIIIII.. PAPPSPSPSP E.TIGDYTWTNGSQPCVHLQAQIQIRVMYTTQGGGE 189 . .::.I. I: .:I:.: I . SPSMKYNVSGTNGTCLLASMGLQLNLTYERKDNTT 250 .:... ...... AWGISVLNPNKTKVQGSCEGAHPHLLLSFPYGHLSFGFMQDLQQKVVYLS 239 .. . : :IIIII..IIIIII I . I : I : VTRLLN1NPNKTSASGSC.GAHLVTLELHSEGTTVLLFQFGMNASSSRFF 299 ..... YMAVEYNVSFPHAAKWTFSAQNASLRDLQAPLGQSFSC.SNSSI1LSPAV 280 . :::.I. :l.l . .I.I.I:III.III.:I.I:.I .:. : : . . I . LQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNAEEHVRVTW 349 HLDLLSLRLQAAQLPHTGVFGQSFSCPSDRSILLPLIIGLILLGLLALVL .. ....... .. .. .. I 1 . : . . . . . . . . : . l . l : l : : I I 1 1 : : I I . . 338 SVNIFKWQAFKVEGGQFGSVEECLLDENSTLIPIAVGGALAGLVLIVL 399 1AFCIIRRRPSA.YQAL 354 1 1 : : I:I. I 11.: IAYLVGRKRSHAGYQTI 416 the CD68 gene structure and tissue macrophage CD68 protein synthesis will resolve these issues. There are nine potential N-glycosylation sites (Asn-X-Ser/ Thr). Interestingly, because of the presence of two of these sites within the repeat motif (B-A‘), there are only seven potential N-linked glycosylation sites in CD68.2. The predicted mass of the polypeptide backbone in clone CD68.1 is only 35 Kd, whereas the observed mass of the mature glycoprotein is 1 10 Kd. The extracellular domain has numerous contiguous runs of serine, threonine, and proline, which could act as sites for attachment for 0-linked carbohydrate.26Thus, the largest contribution to the mass of the mature CD68 comes from both 0- and N-linked carbohydrate; approximately 30% from 0-linked and 30% from N-linked sugars (assuming an average mass/glycan chain of 4 Kd), with only about one third coming from the polypeptide backbone. A search of the National Biomedical Research Foundation protein database2’ disclosed homology to human lamp 1 l 2 and chicken lep10014(Fig 5); both are ubiquitously expressed glycoproteins located in lysosomal membranes that shuttle in vesicles between the lysosome and plasma membrane.I6 Monte Carlo simulation of the alignment of 500 randomly permuted variants of the two sequences gave a mean score 10.4 standard deviations lower than the alignment score computed for CD68 and lampl. The alignment of CD68 with lamp1 (Fig 5 ) shows 26.6% identity and 45% similarity. The extracellular domain of the lamp/lgp family has a bipartite organization, the two domains being divided by an extended proline hinge.28CD68 has this bipartite structure, divided by the proline hinge; 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 to 37 residues apart) and in the lamp family, intradomain disulphide bonds are formed between the first and second and between the third and fourth cysteine^.^^.^^ The equivalent domain of CD68 also has four cysteines, which align with the equivalent cysteines in lamp 1 and lep 100. From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 1612 HOLNESS AND SIMMONS The cytoplasmic tail is conserved, being relatively short (10 residues). In particular, a key tyrosine residue preceded by a small side-group amino acid (alanine or glycine),” identified by in vitro mutagenesis studies to play a dominant role in lysosomal targeting, is conserved. The N-terminal domain of CD68 is unrelated to the equivalent domain of the lamp/lgp family and is unique, having no close homology to any sequences in the National Biomedical Research Foundation database. It is very dense in serine and threonine residues (55% ser thr), characteristic of mucin-like molecules decorated with 0-linked glycan chains. I On the basis of the sequence and domain homologies presented here, CD68 is a new member of the lamp/lgp family of lysosomal/plasma membrane shuttling proteins. In agreement with this conclusion, immunoelectron microscopy studies using CD68 MoAbs have localized the CD68 antigen to endosomal/lysosomal structures.” Immunohistochemical studies show that CD68 is expressed at very low levels in most cell types, but is abundant in macrophages.’-’ CD68 may play a role in endocytosis or lysosomal traffic that is an essential “housekeeping” activity in all cells. This activity may have been expanded to become a dominant part of the specialized function of phagocytic cells such as macrophages. CD68 contains a region rich in serine and threonine residues (>40%) which may bear 0-linked glycan chains and on this basis, CD68 may be considered a mucin-like protein. There are now several examples of mucin-like membrane proteins on hematopoietic cells. These proteins are abundantly decorated with 0-linked sugars and so have a high serine/threonine content in their polypeptide chains, but show no significant homology at the cDNA level. Members of this mucin-like family include: rat and mouse leuk~sialinl’,~~ and its homologue CD43;’ widely distributed pan-hematopoietic glycoproteins of 100 to 120 Kd about two thirds by mass 0linked carbohydrate; CD34, a marker for human bone marrow stem cells,32is a l 15-Kd glycoprotein with a carbohydrate mass of approx 65 Kd, most of which consists of sialylated 0-linked sugars; and the recently reported lymph node high endothelial ligand for L-selectin or GlyCAM- 1.23 It has been proposed that one of the functions of the hematopoietic mucins is to present carbohydrate ligands to sel e ~ t i n sRotary . ~ ~ shadowing electron microscopy has shown that leukosialin has an extended rodlike structure that could protrude above the glycocalyx of the cell, and allow multiple glycan chains to be accessible for binding.34Lamp-I on leukemic cells has recently been shown to bear sialylated fucosylated polylactosaminoglycans that bind to E-selectin.’* In addition, a 100- to 120-Kd glycoprotein on granulocytes with similar properties to CD68 has been identified as a presenter of glycan-ligands to P-selectin on vascular endotheli~m.~’ This glycoprotein is not lamp1 or lamp2 or CD43, and we are currently investigating whether it could be CD68. CD68 is highly expressed in many tumor cell lines: which could allow them to attach to selectins on vascular endothelium, facilitating their dissemination to secondary sites. Macrophages are highly motile cells and many populations are tissue or organ specific. The normal function of CD68 in macrophages may be to bind to tissue- and organ-specific + lectins or selectins, allowing homing of macrophage subsets to particular sites. Additionally, CD68 could allow macrophages to roll over cells as observed for selectin-mediated rolling of neutrophils over vascular endothelium.% The rapid recirculation of CD68 from intracellular sites (endosomes, lysosomes, vesicles) to the plasma membrane may allow macrophages to crawl over selectin bearing substrates or other cells. Alternatively, CD68 may be proteolytically shed from the cell surface; consistent with this, soluble CD68 is found in serum and urine.’ The over-expression of CD68 in macrophages may allow this locomotion to occur at higher rates than in other cell types. The availability of cDNA clones encoding CD68 will allow us to explore its role in both intracellular and extracellular macrophage functions. ACKNOWLEDGMENT We thank Dr Brian Seed (Department of Molecular Biology, Massachusetts General Hospital, Boston) for allowing the initial stages of the cloning of CD68 to be performed in his laboratory; Dr David Mason (Nuffield Department of Pathology, John Radcliffe Hospital, Oxford, UK) for the anti-CD68 antibodies and transfectant staining. REFERENCES 1. Gordon S Biology of the macrophage. J Cell Sci Suppl4267, 1986 2. Gordon S, Fraser I, Nath D, Hughes D, Clarke S Macrophages in tissues and in vitro. Curr Opin Biol4:25, 1992 3. McMichael AJ, Beverley PCL, Cobbold S, Crumpton MJ, Gilks W, Gotch FM, Hogg N, Horton M, Ling N, MacLennan ICM, Mason DY, Milstein C, Spiegelhalter D, Waldmann H (eds): Leukocyte Typing 111. White Cell Differentiation Antigens. Oxford, UK, Oxford, 1987 4. Knapp W, Dorken B, Gilks WR, Rieber EP, Schmidt RE, Stein H, von dem Borne AEGKr (eds): Leukocyte Typing IV. White Cell Differentiation Antigens. Oxford, UK, Oxford, 199I 5. Micklem K, Rigney E, Cordell J, Simmons D, Stross P, Turley H, Seed B, Mason D A human macrophage-associated antigen CD68 detected by six different monoclonal antibodies. Br J Haematol 73: 6, 1989 6. Parwaresgh MR, Radzum HJ, Kriepe H, Hansmann ML, Barth J: Monocyte/macrophage reactive monoclonal antibody Ki-M6 recognizes an intracytoplasmic antigen. Am J Pathol 125:141, 1986 7. Smith MEF, Costa MJ, Weiss SW: Evaluation of CD68 and other histiocytic antigens in angiomatoid malignant fibrous histiocytoma. Am J Surg Pathol 15:757, 1991 8. Wamke RA, Pulford KAF, Pallesen G, Ralfkiaer E, Brown DC,Gatter KC, Mason D Y Diagnosis of myelomonocytic and macrophage neoplasms in routinely processed tissue biopsies with monoclonal antibody KPl. Am J Pathol 135:1089, 1989 9. Pulford KAF, Sipos A, Cordell JL, Stross WP, Mason DY: Distribution of the CD68 macrophage/myeloid associated antigen. Int Immunol2:973, 1990 10. Seed B, Aruffo S Molecular cloning of a CD28 cDNA by a high efficiency COS cell expression system. Proc Natl Acad Sci USA 848573, 1987 11. Saito N, Pulford KAF, Breton-Gonus J, Mason DY, Cramer E M Ultrastructural localisation of the CD68 macrophage-associated antigen in human blood neutrophils and monocytes. Am J Path01 139:1053, 1991 12. Fukuda M, Viitela J, Matteson J, Carlsson SR: Cloning of cDNAs encoding human lysosdmal membrane glycoproteins, h-lamp 1 and h-lamp-2. J Biol Chem 263: 18920, 1988 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. MOLECULAR CLONING OF CD68 13. Chen, JW, Cha Y, Yuksel, KU, Gracy RW, August JT: Isolation and sequencing of a cDNA clone encoding lysosomal membrane glycoprotein mouse LAMP-I. J Biol Chem 26323754, 1988 14. Fambrough DM, Takeyasu K, Lippincott-Schwarz J, Siege1 N R Structure of LEPIOO, a glycoprotein that shuttles between lysosomes and the plasma membrane, deduced from the nucleotide sequence of the encoding cDNA. J Cell Biol 106:61, 1988 15. Zot AS, Fambrough DM: Structure of a gene for the lysosomal glycoprotein (LEP100). J Biol Chem 265:20988, 1990 16. Lippincot-Schwartz J, Fambrough DM: Cycling of the integral membrane glycoprotein, LEPIOO, between plasma membrane and lysosomes: Kinetic and morphologic analysis. Cell 49:669, 1987 17. Howe CL, Granger BL, Hull M, Green SA, Gabel CA, Helenius A, Mellman I: Derived protein sequence, oligosaccharides, and membrane insertion of the 120-kDa lysosomal membrane glycoprotein (lgp120):Identification of a highly conserved family of lysosomal membrane glycoproteins. Proc Natl Acad Sci USA 85:7577, 1988 18. Saitoh 0, Wang WC, Lotan R, Fukuda M: Differential glycosylation and cell surface expression of lysosomal membrane glycoproteins in sublines of a human colon cancer exhibiting distinct metastatic potentials. J Biol Chem 2675700, 1992 19. Killeen N, Barclay AN, Willis AC, Williams A F The sequence of rat leukosialin (W3/13 antigen) reveals a molecule with 0-linked glycosylation of one third of its extracellular amino acids. EMBO J 6:4029, 1987 20. Cyster JC, Somoza N, Killeen N, Williams A F Protein sequence and gene structure for mouse leukosialin (CD43), a T lymphocyte mucin without introns in the coding sequence. Eur J Immunol20875, 1990 2 1. Shelley CS, Remold-ODonnell E, Davis AE 111, Bruns GAP, Rosen FS,Carroll MC, Whitehead MC: Molecular characterisation of sialophorin (CD43), the lymphocyte surface sialoglycoprotein defective in Wiskott-Aldrich syndrome. Proc Natl Acad Sci USA 86: 2889, 1989 22. Simmons DL, SatterthwaiteAB, Tenen DG,Seed B Molecular cloning of a cDNA encoding CD34, a sialomucin of human haematopoietic stem cells. J Immunol 148:267, 1992 23. Lasky LA, Singer MS, Dowbenko D, Imai Y, Henzel WJ, Grimley C, Fennie C, Gillett N, Watson SR, Rosen S D An endothelial 1613 ligand for L-selectin is a novel mucin-like molecule. Cell 69:927, 1992 24. Sanger F, Nicklen S, Coulson A: DNA sequencing with chainterminating inhibitors. Proc Natl Acad Sci USA 743463, 1977 25. von Heijne G: A new method for predicting signal cleavage sites. Nucleic Acids Res 14:4683, 1986 26. Wilson IBH, Gavel Y, von Heijne G Amino acid distributions around 0-linked glycosylation sites. Biochem J 275529, 1991 27. Wilbur WJ, Lipman DJ: Rapid similarity searches of nucleic acid and protein data banks. Proc Natl Acad Sci USA 80:726, 1983 28. Fukuda M: Lysosomal membrane glycoproteins. J Biol Chem 266:21327, 1991 29. Carlsson SR, Fukuda M: Structure of human lysosomal membrane glycoprotein 1. J Biol Chem 264:20526, 1989 30. Arterbum LM, Earles BJ, August JT: The disulfide structure of mouse lysosome-associated membrane protein I. J Biol Chem 26274 19, 1990 3 I. Fukuda M: Leukosialin, a major 0-glycan-containing sialoglycoprotein defining leukocyte differentiation and malignancy. Glycobiology 1:347, 1991 32. Sutherland DR, Watt SM, Dowden G, Karhl K, Baker MA, Greaves MF, Smart JE: Structural and partial amino acid sequence analysis of the human hemopoietic progenitor cell antigen CD34. Leukaemia 2:793, 1988 33. Williams A F Out of equilibrium. Nature 352:473, 1991 34. Cyster JG, Shotton DM, Williams A F The dimensions of the T lymphocyte glycoprotein leukosialin and identification of linear protein epitopes that can be modified by glycosylation. EMBO J IO: 893, 1991 35. Moore KL, Stutts NL, Diaz S, Smith DF, Cummings RD, Varki A, McEver R P Identification of a specific glycoprotein ligand for P selectin (CD62) on myeloid cells. J Cell Biol 118:445, 1992 36. Lawrence MB, Springer TA: Leukocytes roll on a selectin at physiologic flow rates: Distinction from and prerequisite for adhesion through integrins. Cell 655359, 1991 37. Devereux J, Haeberli H, Smithies 0: A complete set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387, 1983 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 1993 81: 1607-1613 Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins CL Holness and DL Simmons Updated information and services can be found at: http://www.bloodjournal.org/content/81/6/1607.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.