Download Molecular Cloning of CD68, a Human Macrophage

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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
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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
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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.
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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.
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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.
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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.
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1993 81: 1607-1613
Molecular cloning of CD68, a human macrophage marker related to
lysosomal glycoproteins
CL Holness and DL Simmons
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