(CANCER RESEARCH 49, 6077-6084. November 1. 1989]

Molecular Characterization of P2B/LAMP-1, a Major Protein Target of aMetastasis-associated Oligosaccharide Structure1

M. Heffernan, S. Yousefi, and J. W. Dennis2

Division of Cancer and Celt Biology, Mount Sinai Hospital Research Institute, Toronto, Ontario M5G 1X5 [M. H., S. Y., J. W. D.], and Department of Medical Genetics,University of Toronto, Toronto, Ontario M5S IA8 [M. H. J. W. D.], Canada

ABSTRACT

Sialylated and G\cNAcßl-6Manal-6Manßl (pil-k branched) complex-type oligosaccharides linked to asparagine residues of membraneglycoproteins in metastatic murine tumor cells have been associated withefficient tumor cell metastasis. A large proportion of these oligosacchar-ide structures, in several unrelated malignant cell lines, have been shownto be associated with a glycoprotein termed P2B, with a molecular weightof 130,000. This glycoprotein has recently been purified from the metastatic MDAY-D2 cell line and shown to be biochemically similar to alysosomal associated membrane glycoprotein (LAMP-1). We report herethe details of a 2147 nucleotide complementary DNA encoding the entiremurine P2B polypeptide which was immunoselected from a Xgtl 1 expression library and sequenced. The sequence is similar to a complementaryDNA coding for mouse LAMP-1 with the exception of a 5' untranslated

region, a leader signal-sequence, and various insertions, deletions, andsubstitutions in the 3' untranslated domain. An open reading frame of

405 amino acids encodes a mature polypeptide of 382 residues with apredicted molecular weight of 42,000. P2B/LAMP-1 possesses 20 as-paragine-linked glycosylation sites separated into equal halves by acentral, putative hinge region and is anchored by a carboxy, membrane-spanning, domain. Topologica! considerations dictate that cell surfaceexpression of P2B/LAMP-1 exposes the bulk of the glycoprotein intothe extracellular compartment. Immunofluorescent staining of fibroblastcells indicated that P2B/LAMP-1 was associated with lysosomal membranes and, to a lesser degree, select surfaces of plasma membrane. Anamino acid comparison of the murine sequence with its recently clonedrat, human, and chicken counterparts shows a conservation of 17 of 20asparagine-linked glycosylation consensus sites, eight of eight cysteineresidues, and other selected protein domains. The interspecies conservation of these domains suggests that they are important for the structureand function of the P2B/LAMP-1 glycoprotein. Northern analysis revealed that P2B/LAMP-1 is widely expressed in normal murine tissuesand tumor cell lines. However, in two experimental models of metastasis,where changes in branching of oligosaccharides on P2B/LAMP-1 havebeen shown to occur, comparable levels of P2B/LAMP-1 mRNA werefound in both metastatic and nonmetastatic cell lines.

INTRODUCTION

Neoplastia transformation is often accompanied by structuralalterations in the oligosaccharide portion of cellular glycoproteins (1-5) and glycolipids (6, 7). Increased ßl-6branching3 atthe trimannosyl core of complex-type ASM-linked oligosaccharides has previously been associated with transformation ofrodent fibroblasts (8, 9) and, more recently, with enhanced

Received 12/1/88; revised 6/7/89; accepted 8/4/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by the National Cancer Institute of Canada (J. W.

D.). J. W. D. is a research scholar of the National Cancer Institute of Canada,and M. H. possesses a Medical Research of Canada studentship. The nucleotidesequence data reported in this paper have been submitted to the GenBank, EMBL,and DDBJ nucleotide sequence databases and conform to IUPAC.

1To whom requests for reprints should be addressed, at Division of Cancer

and Cell Biology. Ml. Sinai Hospital Research Institute. 600 University Avenue,Toronto, Ontario M5G 1X5, Canada.

3The abbreviations used are: rf1-6 branch, GlcN Ac/3/-6Mana l -6Manöl ; ASM,asparagine; LAMP-1, lysosomal associated membrane glycoprotein; L-PHA,leukoagglutinin; cDNA, complementary DNA; TBS, Tris-buffered saline [50 mMTris-HCl (pH 8.0)-150 mM NaCl]; PBS. phosphate-buffered saline; BSA, bovineserum albumin; TPA, 12-0-tetradecanoylphorbol-13-acetate.

metastatic potential in several experimental tumor models (10).In this regard, glycosylation mutants of the metastatic MDAY-D2 lymphoma cell line, which are deficient in ßl-6GlcNAc-transferase V activity, show loss of metastatic potential butretain full tumorigenic potential. Similarly, premature truncation of oligosaccharides due to an apparent deficiency in UDP-galactose transport into the Golgi apparatus results in loss ofmetastatic potential (i.e., Class 1 glycosylation mutants). Finally, inhibitors of asparginine-linked oligosaccharide processing, such as swainsonine and castinospermine, which block thepathway prior to the initiation of the .il 6 linked antenna, alsoinhibit metastasis (11, 12).

The Class 1 glycosylation defect has also been associatedwith increased cell adhesion on surfaces coated with the extracellular matrix proteins fibronectin, laminin, and collagen (13).We have also recently shown that swainsonine-induced inhibition of ASM-linked oligosaccharide processing in metastaticmurine mammary carcinoma cells is associated with increasedtumor cell adhesion to, and decreased invasion of, humanamnion basement membrane in vitro.4 These observations suggest that expression of 01-6 branched complex-type oligosaccharides on the surface of metastatic tumor cells may reducecell adhesion to the extracellular matrix at the primary site ofgrowth and therefore increase tumor invasion and metastasisto distant organs.

The majority of sialylated, /31-6 branched oligosaccharidesobserved ¡nMDAY-D2 cells are associated with glycoproteinsin the M, 80,000- to 160,000-size range, as indicated by lectin

staining of plasma membrane preparations separated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (10). Isolation and characterization of these glycoproteins from MDAY-D2 cells indicated that the majority of ß1-6 branched oligosaccharides were present on two species: a lymphoid-specific glycoprotein P2A (M, 110,000); and a ubiquitous glycoprotein,P2B (M, 130,000) (14, 15). A transformation- and progression-related increase in ßl-6branching observed in rati fibroblastsand in the murine mammary carcinoma cell line, SP1, appearedto be associated with P2B. The glycoprotein is approximately50 to 60% ASM-linked carbohydrate based on monosaccharideanalysis and glycosidase F digestion and was estimated topossess at least 10 to 15 ASM-linked sites. Furthermore, thepurified glycoprotein was found to bind to extracellular matrixproteins with an affinity that is dependent upon its glycosylationstate (15). The presence of sialylated and polylactosamine-containing ASM-linked structures, on purified P2B, reduced itsaffinity for immobilized fibronectin, collagen type I, and laminin, analogous to the cellular phenotypes discussed above. Sincethe glycosylation status of this glycoprotein has been implicatedin metastasis, a cDNA encoding P2B was sought in order tostudy further the relationship between the protein, its carbohydrate, and metastasis.

4 S. Yagel, R. Feinmesser, C. Waghorne, P. Lalo, M. Breitman, and J. Dennis.Evidence that .(16 branched Asn-linked oligosaccharides facilitate tumor cellinvasion of basement membranes by reducing cell adhesion. Int. J. Cancer, inpress. 1989.

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MOLECULAR CHARACTERIZATION OF P2B/LAMP-I

MATERIALS AND METHODS

Isolation of P2B Xgtll Clones. A murine B-eell leukemia cDNAexpression library in Xgtll (16), cell line 7OZ/3 (17) (gift of AndreBernards), was grown on Escherichia coli Y1090 and screened withpolyclonal antiserum raised against purified P2B. The rabbit anti-mouse P2B polyclonal antiserum was prepared against gel-purified P2Bas described (14). Approximately 1 x 10s plaques were plated in LBagarose and incubated at 42°Cfor 4 h. The plates were then overlaid

with nitrocellulose filters (BioRad) previously wetted in 10 mM isopro-pylthiogalactoside (Boehringer Mannheim) and incubated at 37°Cfor

2 h. The filters were washed in TBS and subsequently blocked with20% fetal calf serum in TBS for 30 min. The filters were then incubatedovernight with a 1:500 dilution of polyclonal anti-P2B antiserum thathad previously been absorbed twice with nitrocellulose filters dipped inE. coli lysate Y1090. An affinity-purified goat anti-mouse, alkalinephosphatase-conjugated, IgG detection system was used (BioRad) toidentify antibody binding recombinant phage. Positive phage recombinants were plaque purified (18). The DNA was isolated using a mini-lysate technique (19) and digested with EcoR\ in order to determinethe size and to subclone the cDNA inserts.

Western Analysis. Affinity-purified antibodies specific for putativeclones were prepared using the following method. Phage were platedin LB agarose and grown to confluency in the manner previouslydescribed. Following an overnight incubation with a 1:500 dilution ofanti-P2B polyclonal antiserum, the filters were washed in 3 changes ofTBS plus 1% Nonidet P-40 for 1 h each. Following the removal ofnonspecific antibodies, the filters were incubated in 0.2 M glycine:0.5M NaCI (pH 2.8) for 10 min at 4°C.The solution now containing clone-

specific antibodies was immediately neutralized with l M Tris-HCl (pH7.4), and fetal calf serum was added to a final concentration of 20%.

Purified P2B separated by a 12% sodium dodecyl sulfate-polyacryl-amide gel was transferred electrophoretically onto nitrocellulose in a25 mM Tris-HCl:0.2 M glycine:20% methanol buffer overnight at 4°C.

Following transfer the sheet was blocked with 20% fetal calf serum inTBS. The clone-specific purified antibodies were used to challenge theblot. A negative control of clone-specific antibodies to a putativetyrosine kinase, phage recombinant, was also made. The filters wereincubated with the undiluted antibody solution overnight, and thealkaline detection system (BioRad) was used to detect affinity-purifiedantibodies bound to P2B.

For glycosidase digestion, samples of purified P2B were dissolved in10 mM Tris-HCl (pH 7.5): 150 mM NaCl:0.4% deoxycholate:l% TritonX-100 for digestions with 1 unit of glycopeptidase F (BoehringerMannheim) overnight at 37°C.Digestions of P2B with endo-/3-galac-tosidase (Miles) were performed overnight at 37°Cin 10 mM Tris-HCl

(pH 7.5): 150 mM NaCl:l% Triton X-100:50 mM sodium acetate (pH5.5):5 inillinnil-, of enzyme.

Subcloning and Sequencing. Four putative positive clones identifiedin the Western experiment were subcloned in the EcoRl site ofpBluescript KS (Stratagene Cloning Systems, La Jolla, California).Deletion clones were constructed using the £jtoIII/Mung Bean nucleasedeletion system (Stratagene Cloning Systems). Relevant fragments ofthe cDNA, not covered by the deletions, were subcloned to facilitatethe sequencing of the full 2147 base pairs of the clone. Single-strandedDNA was isolated and sequenced according to the Sanger dideoxymethod (20) and the enzyme Sequenase (United States Biochemical).Areas of sequence overlap between subclones were at least 50 base pairsin length. Both strands of the DNA were sequenced in segments of thecDNA that were novel. Sequence analysis was performed with softwarefrom the Genetics Computer Group, University of Wisconsin (21).

Northern Analysis. Total RNA was isolated from MDAY-D2 andD36W25 using the isothiocyanate/guanidinium method (22). TissueRNA was isolated using the method of Chomczynski and Sacchi (23).RNA isolated from the cell lines SP1, SPl-T24rasl, SP1-A3 (calciumionophore treated), SP1-T1 (TPA treated), and SP1-M1 (calcium io-nophore plus TPA treated) was obtained from Dr. Korczack. RNAisolated from rat2-neo, rat2-neo/T24ras, and rat2-neo/T24ras/c-mycwas obtained from Dr. Brietman. Total RNA was electrophoresedthrough a formaldehyde-agarose gel and transferred overnight (22) ontoa Zeta Probe membrane (BioRad) in 50 mM NaOH. A riboprobe

(Promega) or random primed probe (Boehringer Mannheim) constituting a portion of the cDNA, excluding the polyadenylate tail, was usedto probe the membrane.

Immunofluorescent Localization. Rat2 cells were grown on glasscoverslips for 48 h, washed with PBS, and were fixed and permeabilizedwith ice-cold acetone for 3 to 5 min. The coverslips were air dried andadded to a solution of PBS/0.1 % BSA containing either a 1/10 dilutionof rat anti-P2B monoclonal antibody, a 1/200 dilution of rabbit anti-P2B polyclonal antiserum, or 2 ng/ml of L-PHA for 1 h at 20°C.Inthe case of L-PHA staining, the coverslips were washed 3 times, 5 mineach in PBS/0.1% BSA, and incubated for an additional 1 h in a 1/500dilution of rabbit anti-L-PHA. The coverslips were washed 3 times, 5min each, with PBS/0.1% BSA and then incubated for another hourwith either a 1/100 dilution of goat anti-rat fluorescein isothiocyanate-conjugated IgG (Sigma) or 1/100 dilution of goat anti-rabbit fluoresceinisothiocyanate-conjugated IgG (Sigma).

Stained, nonpermeabilized rat2 cells were first grown on glass cover-slips for 48 h as above. Cells were washed twice with PBS and incubatedwith shaking for 45 min in 1% gluteraldehyde:PBS:0.1% BSA. Following removal of this solution, the cells were incubated twice, for 4 mineach, in 0.5 mg/ml of sodium borohydride. The coverslips were airdried and incubated with the anti-P2B polyclonal antiserum as outlinedabove.

RESULTS

Isolation of P2B cDNA. Since the rabbit anti-P2B antiserumraised against the native glycoprotein, reacted with glycopeptidase F-treated P2B in Western blots (i.e., deglycosylated) (14),it appeared to be suitable for screening an expression library inE. coli. A Xgtl 1 cDNA expression library, prepared from pre-B-cell line 7OZ/3 (16), was screened with the antiserum, andof the approximately 4x10" plaques that were plated, fourputative positive clones were detected. Since MDAY-D2 is alymphoreticular cell line (24), an expression library of the samecell lineage was used. The size of the EcoRl inserts in the uniquecloning site of Xgtl 1, from the positive plaques, was determinedby agarose gel electrophoresis to be on the order of 2.1 kilobases(data not shown). The size of these inserts was approximatelytwice that required to code for the M, 39,000 to 40,000 proteincore of P2B.

The antigenic relationship between the proteins produced bythe selected Xgtl 1 clones and purified P2B was confirmed priorto sequencing the inserts. Affinity-purified antibodies, specificto each of the recombinant phage clones, were isolated. Theseantibody preparations were then tested for their reactivity withpurified P2B in its natural state, lacking polylactosamine antennae, and devoid of ASM-linked oligosaccharides, throughWestern analysis (Fig. 1). All four affinity-purified antibodypreparations bound to native, endo-0-galactosidase, and glycopeptidase F-treated P2B in Western blots. The signal producedincreased as the carbohydrate content of the glycoprotein decreased. This is not surprising, since the recombinant phageproduct, synthesized in a bacterial environment, is not glyco-sylated. As a result, the affinity-purified antibodies were selectedfor specificities reactive against protein epitopes only. Glycosidase digestion of P2B also reduced size heterogeneity, resultingin concentration on the Western blot and thereby contributingto the increased signal. The remaining three positive clonesgave similar results (data not shown). Affinity-purified antibodies selected from the same stock of polyclonal antiserum usinga clone with an unrelated insert, showed only a weak signalwith purified, glycopeptidase F-treated P2B and no signal withP2B in its native, glycosylated state.

Complete Coding Sequence and Species Homology for P2B/LAMP-1. An EcoRl insert from one of the recombinant phage

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MOLECULAR CHARACTERIZATION OF P2B/LAMP-1

3 4l

^-130

—¿�75

—¿�50

—¿�39

-+-27

Fig. l . Western blot of native and exoglycosidase-treated samples of purifiedP2B using affinity-purified antibodies obtained from plaque hybridization with apositive (Lanes 3 to 5) and negative (Lanes 1 and 2) Xgtll clone. Relativemolecular weights are indicated on the right. Lanes I and 5, native P2B (10 /ig);Lanes 2 and 3, glycopeptidase F-treated P2B (10 ^g): Lane 4, endo-0-galactosid-ase-treated P2B (10 ng).

clones was subcloned into pBluescript KS and sequenced (Fig.2). The cDNA is 2147 base pairs in length, of which 112 basepairs represent a 5' untranslated region, 1215 nucleotides en

code a protein of 405 amino acids, and 819 base pairs aresituated in the 3' untranslated region. The entire coding sequence is identical to murine LAMP-1 with the exception ofthe first 69 nucleotides representing the amino-terminal 23amino acids. Within this region is encoded the initiator methi-onine and, as expected for an integral membrane glycoprotein,a hydrophobic leader sequence. The composition of the leaderis in accordance with the observed elements contained withinknown leaders (25). There is a basic TV-terminal region, containing two Arg residues, followed by a hydrophobic centralcore consisting of five Leu residues, and following this, a morepolar C-terminal region. The site of cleavage follows the "(-3,-l)-rule" to yield leucine as the first amino acid in the mature

glycoprotein (26).The mature polypeptide deduced from the cDNA sequence

is 382 amino acids, with a predicted molecular weight of 4 1,978.This is in agreement with the size of P2B/LAMP-1 treatedwith glycopeptidase F to produce a core polypeptide with amolecular weight of 39,000 to 40,000 (14). Two halves of thepolypeptide are separated by a putative hinge region rich inproline, Serine, and threonine residues. Contained within eachhalf is an abundance of ASM-linked glycosylation sites (Asn-X-Ser or Asn-X-Thr), 10 per half. A sequence comparison of

this glycoprotein across four species —¿�mouse, rat (27), human

(28), and chicken (29) —¿�reveals that 11 ASM-linked glycosyla-tion sites are conserved in any three species within a spacing of2 amino acids (Fig. 3). Furthermore seven of these sites areabsolutely conserved in position and spacing across all four

_. . . ., ., .,.,.,,Species. Eight Cysteme residues reside Within the full sequence,four per half. The location of all 8 residues is conserved abso-

lntelv in T AMP-1 apro«;«;the four «¡nerie«;mentioned above ÕFielUteiy m L Ami I across ine lour species mentioned aoove >rig.

3). The spacing of amino acids between each pair of consecutive

Cysteine residues in the protein is similar (38, 35, 35, and 36

amino acids); however, no internal homology between the domains was observed. Amino acid sequence homology of themature polypeptide, between mouse and rat, mouse and human,and mouse and chicken, was found to be 83%, 67%, and 50%,respectively.

The carboxy terminus of the glycoprotein contains a hydro-

-112 GA ATTCCCCGTGCCGGCACCGCAGCTGCGGCGTCTCGAGTCGCTGGA -66

-65 CGCGCCCTCCCGCCGGCCTCGCCCCCCGCGCAACCCCCGTCCTCCGGCCTCGGCTGCGTCGCGCC-1

1 ATCCœaXCCGCGCGŒGCGGCCGTCCTCCTGCTGCTGCTG OCA GGC CTT OCA 5«1 net Arg Pro Pro Arg Ala Ala Ala Val Leu Leu Leu Leu Leu Ala Gly Leu Ala 18

55 CAT GGC GCC TCA OCA CTC TTT GAGGTC AAAAAC AAT OGC ACA ACG TCT ATA ATG 10819 His Gly Ala Ser Ala Leu Phe Glu Val Lys Afin Afin Gly Thr Ttir Cy«lie Het 36

109 GCC AGCTTCTCTGCCTCCTTTCTGACCACCTAC GAGACT GCG AAT OGT TCT CAG 16237 Ala Ser Phe Ser Ala Ser Phe Leu Thr Thr Tyr Glu Thr Ala Aan Gly Ser Gin 54

163 ATC GTG AAC ATT TCC CTG CCA GCC TCT GCAGAAGTA CTG AAAAAT GGCACT TCT 21655 lie val Asn lie Ser Leu Pro Ala Ser Ala Glu Val Leu Lyi Asn Gly Ser Ser 72

217 TOT GGT AAAGAAAÀTGTT TCT GAC CCC AGC CTC ACÕATT ACT TTT GGAAGAGGA 27073 Cy»Gly Ly»Glu Asn val Ser Asp Pro Ser Leu Thr He Thr Phe Gly Arg Gly 90

271 TAT TTA CTG ACA CTC AÂCTTC ACA AAAAÂTACA ACA CCT TAC AGT GTC CAO CAT 32«91 Tyr Leu Leu Thr Leu Asn Phe Thr Lyi Asn Thr Thr Arg Tyr S«rVal Gin Hit 108

325 ATG TAT TTT ACA TAT AAC TTG TCA GAT ACA GAACAT TTT CCC AAT GCC ATC AGC 378109 Net Tyr Phe Thr Tyr Asn Leu Ser Asp Thr Glu Hl»Phe Pro Asn Ala Ile Ser 126

379 AAAGAGATC TAC ACC ATG GAT TCC ACA ACT GAC ATC AAGGCA GAC ATC AAC AAA <32127 Lys Glu He Tyr Thr Met Asp Ser Thr Thr Asp Ile Lys Ala Asp Ile Asn Lys 1(4

433 GCA TAC COG TGT GTC AGT GAT ATC CGG GTC TAC ATG AAGAÂTGTG ACC GTT GTG 466145 Ala Tyr Arg Cys Val S»rAsp Ile Arg val Tyr Bet Ly»Asn Val Thr Val Val 162

487 CTC CGGGAT GCC ACT ATC CAG GCC TAC CTG TCO AGT GGC AAC TTC AGC AAGGAA 540163 Leu Arg Asp Ala Thr Ile Gin Ala Tyr Leu Ser Ser Gly Asn Phe Ser Lys Glu 180

541 GAGACA CAC TGC ACA CAG GAT GGACCT TCC CCA ACC ACT GGGCCA CCC AGC CCC 594181 Glu Thr His Cys Thr Gin Asp Gly Pro Ser Pro Thr^JThr Gly Pro_£ro Ser ^ro 198

595 TCA CCA CCA CTT GTG CCC ACA AÂCCCC ACT GTA TCC AAGTAC AÂTGTT ACT GGT 648199 Ser Pro_Pro Leu val^ Pro Thr Asn Pro Tti£val Ser Lys Tyr Asn Val Thr Gly 216

619 AAC AAC GGAACC TGCCTGCTGGCCTCTATGGCACTGCAACTG AAT ATC ACC TAC 702;i^ Asn Asn Gly Thr Cys Leu Leu Ala Ser net Ala Leu Gin Leu Asn Ile Thr Tyr 234

-03 CTG AAAAAGGAC AAC AAGACG GTG ACC AGAGCG TTC AAC ATC AGC CCA AAT GAC 756235 Leu Lys Lys Asp Asn Lys Thr Val Thr Arg Ala Phe Asn Ile Ser Pro Asn Asp 252

757 ACA TCT ACT GGGAGT TGC GGT ATC AAC TTG GTG ACC CTG AAAGTG GAGAAC AAG 810253 Thr Ser Ser Gly Ser Cys Gly Ile Asn Leu Val Thr Leu Lys Val Glu Asn Lys 270

811 AAC AGAGCC CTG GAA TTG CAG TTT GGGATG AAT GCC AGC TCT ACC CTG TTT TTC 864271 Asn Arg Ala Leu Glu Leu Gin Phe Gly «et Asn Ala Ser Ser Ser Leu Phe Phe 288

8*5 TTG CAA GGAGTG CGC TTG AAT ATG ACT CTT CCT GAT GCC CTA GTG CCC ACA TTC 918289 Leu Gin Gly Val Arg Leu Asn net Thr Leu Pro Asp Ala Leu Val Pro Ttir Phe 306

919 AGC ATC TCC AAC CAT TCA CTG AAAGCT CTT CAGGCC ACT GTG GGAAAC TCA TAC 972307 Ser Ile Ser Asn His Ser Leu Lys Ala Leu Gin Ala Thr Val Gly Asn Ser Tyr 324

973 AAG TGC AAC ACT GAGGAACAC ATC TTT GTC AGC AAGATG CTC TCC CTC AAT GTC 1026325 Lys Cys Asn Thr Glu Glu His Ile Phe Val Ser Lys net Leu Ser Leu Asn Val 342

1027 TTC AGT GTG CAG GTC CAG GCT TTC AAGGTG GACAGT GACAGOTTT GGGTCT GTG 1080343 Phe S«rVal Gin Val Gin Ala Phe Lys Val Asp Ser Asp Arg Phe Gly Ser Val 360

1081 GAAGAGTCT GTT CAG GAT GGTAAC AAC ATGITTG ATC CCC ATT GCT GTG GGC GGT 1134361 Glu Gl-j Cys Val Gin Asp Gly Asn Asn net [Leu He Pro Ile Ala Val Cly Gly 378

AGOAAG 1188113: GCC CTG GCA GGGl379 Ala Leu Ala Gly Leu Ile Leu Ile Val Leu Ile Ala Tyr Leu Ile Gly Arg Lys 396

1189 AGGAGT CAC GCC GGC TAT CAGACC ATC TAG CCTGGTGGGCAGGTGCACCAGAGATGCACAG1248397 Arg Ser His Ala Gly Tyr Gin Thr Ile End

1249 C«XXlVnVlCACATCCCCaACOTAaTACGTCTCC*AGGGACCOlC^^ 1319

1320 MTCTGCITTATCAAATGTGAACnTCATCTTGCAACATTTACTATCOkCAAAGGM 1390

1391 CGCTCTTAATTTTGCTAACTGarrouWTATTrTC<riWuri^ 1461

1462 GCTCTAAAGAGGATGACJiACGCTCCACTGATTTCAaTmAGAlTIU^^^ 1532

1533 CTCAGATTTAAGCCTTACAAAGGGAATCTCTÅ“TCCAGArCCTTGGGCCTGGO 1603

1604 GGCTGCACACTTAAGAAGCAAACGCWG<>Ga»AGCCITGCCACACA^ 1674

1675 G*CAL'n^jC<Tl'jGGCrACCTGGCL"ri\jC<XXXKXrnlAA¿'lvl\XjCAiCl<»C<?TTjGGTACACACCCCCCAA1745

1746 rnVllTGCrCTCCCACCCCTGACCTGCCACTTTCCTAAATAGAAAATaX^ 1816

1817 TGTAAAGTGAlTlCCAO'lV'l'iVltjI'lVjGCblnt^CCCTGGCCC'iTj'lVlVlVjCAL'lljlü'lACAATAATAGAT1887

1888 TCACACTGCTXjACujwlvi-luCAGCC^IÄGCnXÄjGTTGTACACTGGC^^TCAGCTCACGTAATGCATTGCCT1958

1959 CmACGATCCTAATAAAAACTCTCmvmvlV/MUiAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA2029

2030 AAAAA 2034

Fig. 2. Nucleotide sequence and deduced amino acid sequence of mouse P2B/LAMP-1. Nucleotide residues are numbered in the 5'- to 3'-direction beginningwith the first residue of the translation codon ATG, and nucleotides 5' to this

triplet are indicated by negative numbers. The putative signal peptide is indicatedby a dark avertine, the carboxy membrane-spanning domain is boxed, and theputative hinge region is marked with a dashed underline. The location of the 20ASM-linked glycosylation sites is indicated with asterisks.

phobic anchor sequence of 24 amino acids capable of spanninga membrane (30), followed by three positively charged stoptransfer residues, Arg, Lys, Arg (30). A comparison of thisanchor sequence in the mouse, rat, human, and chicken showsa near perfect conservation (Fig. 3). Amino acids differing from

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MOLECULAR CHARACTERIZATION OF P2B/LAMP-I

the conserved sequence, present in any of the other threespecies, are substitutions maintaining the hydrophobic natureof this segment.

Particular segments of the P2B/LAMP-1 protein sequence,possessing no known biological significance, are strongly conserved across all four species (Fig. 3). Notably, the cytoplasmicdomain following the anchor, representing the final 11 residuesat the C-terminus of the glycoprotein, is absolutely conservedacross all four species. In addition, an 80% amino acid similarity exists over a span of 24 residues following the putative hingeregion. Other well-conserved pockets of amino acids, shorter inlength, also exist in the amino and carboxy portions of thepolypeptide.

Contained within the 3' untranslated region is the polyaden-

ylate addition site 19 nucleotides upstream from the additionof the polyadenylate tail. In addition, sequences present in thiscDNA of LAMP-1, in the 3' untranslated region, differ from

that published recently (31). There is a 50-base pair insertionat residue 1422 (Fig. 2) containing a Sad restriction enzymesite, present also in the human and rat homologues, which isnot found in the LAMP-1 cDNA published by Chen et al. (31).Furthermore, there are 1- and 2-base pair insertions, deletions,and substitutions directly downstream of the 50-base pair insertion. These differences are likely the result of a polymorphism between mouse strains used to generate the respectivelibraries. Apart from these differences, this region is identical

O v 49LAMP-1 MRPPRAAA VLL1.LLAGLA HGASALFEVK .NNGTTCIMA SFSASFLTTY

Lgpl20 MAAPGARRP LLLLLGAGLA HSAPALFEVK DNNGTACIMA SFSASFLTTYLAMP-a MA RGGRVRCIMA NFSAAFSVNY

LeplOO MGG AARAVLLGFL QASSSFDVRD .STGKVCIIA NLTVAFSVEY

LAMP1Lgpl20I.AMP-a

LeplOOLAMP-1Lgpl20LAMP-aLeplOOLAMP-1Lgpl20LAMP

aLeplOOLAMP

1Lgpl20LAHP-aLeplOOLAMP-1Lgpl20LAMP

aLeplOOLAMP

1Lgpl20LAMP-aLeplOOLAMP-1Lg[-l?0LAMP-aLeplOOLAMP-1Lgpl20LAMP-aLeplOO50ETANG

SOIVBDAGHVSKV^jDTKSGPKftiT

KSSGOKOFAH100TKRTTRYSVOTKNTTRYSVOTRMATRYSVOSKTLDKYOVE150CVSDIRVYMKCVSDIRVYMKCVSGTOVHHNCINSKYVRHK*200ppppppAPTTPKHATS250ITYLKKnfkTITYMKKDflrTLTYERKDJfTTTTYVKKDEKM300HlkSSSLFFLMWTSSLFFLMMASSSRFFLLWSSFKFFL350EEHIFVSKMLEEHIFVSKALEEHVRVTKAFEENFOVTDKA400ALAGLI

LIVLALAGLVLIVLALAGLVLIVLALAGLVLIVLISLPASAF.VLMTLPASAEVLFDLPSDATVV

FFLPONA.TSHMYFTYi*.SDHMYFTYM.SDLMSFVÕNLSDELTFHiyLSDINfrrvvLRDATMVTIVLUDATbjiTVTLHDATHVfllTFSNVTSPSPPSPSPPSPSPSOVPTTSPAPTVTRAFNISP8VTRAFN.lft'VTRLLNINffiGLDLLNFIPHOGVRlRlTLPOGVOLJflTLPOCIOLNTILPOGVQVSTTLPSI.NVFSVOVOALNVFSVOVOSVNIFKWVOLVNVFNVOVOIAYLIGRKRSIAYLIGRKRSlAYLVGRKRSIAYLIGRKRSfSSSCGKERSSCGEKNOSHSSCr.EGMTEHFPNAISKTOFFPffcSSKTHLFPHSSSKETLFPJJATECIOAYLSSG»IOAYLPSSBFlOAYL^SSFLEAYPTtfJTFLVPTNPTVSKLVPTNPSVSKPVPKSPSVDKAAPSSPAVCKfDTS.SGSCGISDKYSGTCGAKTSASGSCGAOTSASGMCES.DALVPTFSI.DAIEPTFST.DARDPAFKASEAKAPTFEAAFKVDSDRFGAFRVESDRFGAFKVEGGOFGAFKVDGDKFC427HACYOTI*HAGYOTI*HAGYOTI*HAGYOTI*VSDPSLTITFASEPTLAITFTSDPSLVIAFTSHPUDLSFEIYTMDSTTDCPDTVDSTTDEIKTVESITDKVMVATOKSVTSKEETHCTODSKEETRCPODSRCETRCEODSANKTFÇRED1YM/TflTJlTf^n^^pjL^P•

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GAGHLISLtf149IKADINKAYRIKADINKTYRIRADIDKKYRlOARJGTEYR199GPSPTT...GOPSPTT...GRPSPTTAPPAMVSTTT

...V249LLASMALOLRl.LASMALOlWLLASMGLOÃŽWVLASMGLQlH

•¿�•¿�••...L¿299KNRALELOFGKSRVLELOFGGTTVLLFQFGEKTKITFHFV349TVGNSYKCNTSVGNSYKCNSTVGNSYKCNATVGNSYKCSA399NMLIPIAVGGNMLIPIAVGGSTLIPIAVGGNMLIPIIVGA

Fig. 3. Amino acid sequence similarity between LAMP-1 glycoproteins fromthe mouse, rat (Igp 120) (27), human (LAMP-a) (28), and chicken (LeplOO) (29).Alignment was performed using the LINEUP program. Genetics ComputerGroup, University of Wisconsin (21). Seventeen ASM-linked glycosylation sitesconserved within a spacing of 2 amino acids are boxed. Absolutely conservedcysteine residues are indicated with arrows. All other amino acids conservedacross all four species are marked (*). while gaps in the sequences are indicated

to that found by Chen et al. (31).Northern Analysis. P2B/LAMP-1 has previously been de

tected by Western blotting in the lung, liver, brain, kidney,spleen, intestine, and heart (14). Northern analysis showedhigher levels of P2B/LAMP-1 mRNA in the testes, kidney,liver, and intestine; intermediate levels in the spleen; and lowerlevels in the brain and salivary gland (Fig. 4B). Acquisition ofmetastatic potential is associated with increased branching ofoligosaccharides on P2B/LAMP-1, while the level of the glycoprotein does not appear to be altered. To confirm the latter,Northern analysis was performed to compare P2B/LAMP-1message levels in metastatic and nonmetastatic paired cell lines.As seen in Fig. 4A, the levels of the transcript in MDAY-D2(24) and of its nonmetastatic, Class I glycosylation mutantD36W25 (32) are equal with an approximate message size of2.2 kilobases. This is consistent with Western analyses performed previously, which showed similar P2B protein levels inthese cell lines (14). Similarly, P2B/LAMP-1 message in thetumorigenic but nonmetastatic mammary carcinoma cell lineSP1, and its metastatic, activated H-ros-bearing counterpartSPl-T24rail, revealed no difference in expression (Fig. 4C).Clonal sublines of SP1 which have acquired a stable metastaticcapability following treatment with calcium ionophore, TP A,or both show no increase in P2B/LAMP-1 mRNA levels relative to SP1 (Fig. 4C). However, P2B/LAMP-1 mRNA levelsin the nontumorigenic cell line rat2-neo appeared to be greaterthan that found in the tumorigenic transfectants rat2-T24rasand rat2-T24ras/c-wj'c (Fig. 4D). This cross-species Northernrevealed a decrease in P2B/LAMP-1 message levels in rat2fibroblast cells expressing activated H-ras, and a further decrease in cell lines expressing H-ras and c-myc constitutively.Both the metastatic sublines of SP1 and the tumorigenic transfectants of rat2 have been previously shown to express increased£fl-6branched oligosaccharides on P2B/LAMP-1.

Immunofluorescent Localization. Immunofluorescent localization of P2B/LAMP-1 was performed on subconfluent cultures of rat2 fibroblast cells using polyclonal or monoclonalanti-P2B/LAMP-l antibodies followed by fluoresceinated goatanti-rat or rabbit IgG. The antiserum reacted with vesiclessurrounding the nucleus producing a pattern indicative of ly-sosomal staining (Fig. 5, A and B) and identical to that previously reported for monoclonal anti-LAMP-1 staining in NIH3T3 cells (33). Permeabilized rat2 cells stained with L-PHA, alectin specific for 01-6 branched lactosamine-containing oligosaccharides, show a very similar pattern of perinuclear stainingconsistent with colocalization of P2B/LAMP-1 and the majority of cellular, branched ASM-linked oligosaccharides (Fig. 5C).P2B/LAMP-1 was also expressed on the surface of some, butnot all, rat 2 fibroblast cells. This was confirmed by stainingnonpermeabilized cells which displayed reactivity at surfaceswhich appeared to be retracting edges of motile cells (Fig. 5D).

DISCUSSION

The relationship between expression of 01-6 branched oligosaccharides and metastatic potential (10), as well as theobservation that a large proportion of these structures wereassociated with P2B (gpl30), prompted us to isolate and characterize this glycoprotein (14, 15). Rabbit polyclonal antiserumraised against P2B, purified from metastatic MDAY-D2 cells,was used to screen a Xgtll cDNA expression library. Fourphage bearing identical 2.1-kilobase cDNA inserts were isolated, and their antigenic similarity to purified P2B was confirmed. Sequencing of one of the clones (Fig. 2) revealed an

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Fig. 4. Northern analysis of P2B/LAMP-1transcripts in murine tissues, paired metastaticand nonmetastatic cell lines, and tumorigenicand nontumorigenic cell lines.. 1. total cellularRNA (10 ^g) from the highly metastatic cellline MDAY-D2 (Lane I ) and its nonmetastaticglycosylation mutant, D36W25 (Lane 2). B,total RNA ( 10 fig) from various murine tissues;Lanes 1 to 7 represent brain (Lane I), salivarygland (Lane 2), kidney (Lane 3), intestine(Lane 4), spleen (Lane S), liver (Lane 6), andtestes (Lane 7). C, total RNA from the tumorigenic but nonmetastatic cell line SPI (LaneI), and its metastatic derivatives SPl-T24rail(Lane 2), SP1-A3 (Lane 3), SP1-T1 (Lane 4),and SP1-M1 (Lane 5). D, total RNA from thenontumorigenic cell line rat2-neo (Lane I) andits tumorigenic and metastatic transfectantsrat2-neo/T24rasl (Lane 2} and rat2-neo/T24rasl/c-m>'c (Lane 3). In all of the experiments, the size of the message is approximately2.2 kilobases, as indicated by the rRNA markers.

t* **

-28sB

1234567

•¿�•¿�

-188

12345

D1 2 3

open reading frame identical to that recently reported for murine LAMP-1 glycoprotein (34).

We previously reported the amino acid sequence of the N-terminus of purified P2B as Asp-Phe-GIu-Val-Lys-Asn-Asp-Gly-Thr-Thr-Thr-Ile-Met-Ala-Ser-Phe-Cys-Ala-Met-Phe (10).A comparison of this sequence with that deduced from thecDNA for P2B/LAMP-1 showed five residue differences in thefirst 20 amino acids (Leu-1, Asn-7, Cys-11, Ser-17, Ser-19).Since the alignment of the P2B and LAMP-1 amino endsequences was correct, the discrepant amino acids were likelydue to protein sequencing errors for P2B.

The deduced amino acid sequence encoded in the cDNA ofP2B/LAMP-1 and homology with LAMP-1 sequences of threeother species revealed several interesting structural features(Fig. 3). Of the 405 amino acids encoded in the mRNA, 382are present in the mature polypeptide. The first 23 amino-

terminal residues constitute a leader sequence which was notfound on the mature protein. This sequence is presumablyremoved after facilitating the entry of the nascent protein intothe lumen of the endoplasmic reticulum. Neither an initiatormethionine, nor an amino-terminal leader sequence has beenfound in previous isolates of murine LAMP-1. This hydropho-

bic segment has a 52% and 33% similarity to the 24 amino acidrat LAMP-1 (Igpl20) leader sequence (27) and 18 residuechicken LAMP-1 (Lep-100) leader sequence (29), respectively.Interestingly, only an 18% similarity exists between the murineand proposed human leader sequences of LAMP-1 (28). Thefirst 17 residues of human LAMP-a, considered to be a leadersequence on the basis of its hydrophobic character and residueadherence to the "(-3,-l)-rule" (26), contain a highly conserved

4-amino acid segment at positions 9 through 12 of the murine

sequence. This stretch is present in the mature polypeptide ofmouse, rat, and chicken LAMP-1 and contains one of the 8absolutely conserved cysteine residues in the polypeptide. It islikely that this residue, along with the other cysteines, is important to the tertiary structure of the mature glycoprotein.Further, an alignment of the four species' sequences in Fig. 3

shows an absence of a strong hydrophobic domain at the amino-terminal region of human LAMP-1 which is present in themouse, rat, and chicken sequences. Therefore, it is likely thatthe human leader sequence has not yet been identified.

In all four species, a putative hinge region separates theLAMP-1 protein into approximately equal halves. Thisproline-, serine-, and threonine-rich area is well conservedacross the mouse, rat, and human; however, chicken LAMP-1includes an additional 13 amino acids in the middle of thisregion. In support of this interpretation, the domain also showsa distinct homology with IgAa,-chain (34), Epstein-Barr virus(35), and human lymphotropic virus (36) type II hinge structures (28).

Eight cysteine residues are contained within the murine polypeptide that is absolutely conserved in number and spacingacross rat, human, and chicken forms of LAMP-1. The approximately equal spacing between each pair of consecutive cysteineresidues (38, 35, 35, and 36 amino acids) would seem to suggestthat four loop domains are formed as a result of disulfidebridging. Further, the immobile positions of these residueswould also seem to indicate that such structures are functionallyimportant to P2B/LAMP-1.

Murine LAMP-1 contains 20 potential ASM-linked glycosylation sites, consistent with previous predictions of at least10 to 15 ASM-linked oligosaccharides per molecule (14). The

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D

Fig. 5. Fluorescent labeling of permeabilized rat2 fibroblast cells (A and B) with anti-P2B/LAMP-l antibodies. The cells of A have been stained with a ratmonoclonal antibody raised against purified P2B. while the cells in B have been challenged with rabbit anti-P2B polyclonal antiserum also raised against the purifiedglycoprotein. The intense perinuclear ring is characteristic of lysosomal localization and, to a lesser degree, cell surface localization of P2B/LAMP-1 can also be seenon select cell surfaces in A and B. The permeabilized rat2 fibroblast cells of C were incubated in L-PHA followed by staining with anti-L-PHA antibodies. Cell surfacelocalization of P2B/LAMP-1 was confirmed by staining nonpermeabilized rat2 fibroblast cells with the rabbit anti-P2B polyclonal antiserum (D).

central hinge domain serves to separate these sites in half withrespect to the polypeptide. As shown in Fig. 3, 17 of the 20murine ASM-linked sites are conserved within a spacing of 2amino acids across 4 species, and 7 of the sites are absolutelyconserved. P2B/LAMP-1, isolated from MDAY-D2, was foundto be considerably more resistant to protease digestion in aglycosylated form, in contrast to its sensitivity following gly-cosidase F digestion.5 The oligosaccharides may serve to protect

the glycoprotein from degradation by the various proteasespresent in lysosomes. A multitude of glycosylation sites arepresent in three of the four predicted loop domains; however,the loop closest to the carboxy end is devoid of ASM-linkedsites in all four species. If oligosaccharides were present in thisregion of the glycoprotein, they could interfere with the membrane-anchoring function of the nearby hydrophobic carboxy

domain.A comparison of the LAMP-1 amino acid sequences across

four species revealed highly conserved protein segments in theglycoprotein. Following the hydrophobic membrane-anchoringdomain, at the C-terminus of the glycoprotein, is an 11-aminoacid sequence which is absolutely conserved in the mouse, rat,human, and chicken. This cytoplasmic segment is primarilyhydrophilic in nature, and a search of the National BiomedicaiFoundation Protein databank did not reveal significant se-

' Unpublished observation.

quence similarities to any additional proteins. A 24-amino aciddomain following the putative hinge region has an 80% sequence similarity across four species and contains three of theseven absolutely conserved asparginine-linked glycosylationsites and one of the common cysteine residues. In addition,other regions of high amino acid conservation, but shorter inlength, are distributed throughout the glycoprotein. It will beinteresting to determine whether these regions of cross-specieshomology are required for LAMP-1 function, in particularbinding to extracellular matrix proteins.

Quantitation of P2B/LAMP-1 mRNA by Northern analysis(Fig. 4) and total cellular P2B/LAMP-1 protein by Westernblotting (14) showed no differences when metastatic and non-metastatic paired cell lines were compared. This confirmedearlier suggestions that differences in L-PHA binding to P2B/LAMP-1 were due to a tumor-progression-related increase inbranching of oligosaccharides rather than differences in theglycoprotein levels (10). However, mRNA levels appeared tovary among the seven different normal tissues examined. Interestingly, P2B/LAMP-1 mRNA levels appeared to decrease inoncogene-transfected rat2 fibroblast cells (Fig. 4D). Furtherstudies are being performed in order to determine if this traitis common in other cell lines transfected with dominant-actingoncogenes. The murine tissue Northern confirmed previousWestern analyses (14), indicating that P2B/LAMP-1 is distributed in a variety of tissues. The ubiquitous nature of P2B/

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LAMP-1 suggests that transformation or progression-relatedchanges in oligosaccharide processing on the glycoprotein maybe a common feature of malignancies in a wide range of tissues.

Topological considerations of the sequence indicate that 90%of the glycoprotein faces the luminal membrane side of theendoplasmic reticulum, lysosomal compartments, and extracellular side of the plasma membrane. A highly conserved, 24-amino acid, hydrophobic domain capable of spanning the membrane (30), serves to anchor the glycoprotein in the membraneslisted above. Immunofluorescent staining of rat2 fibroblastswith anti-P2B/LAMP-l polyclonal serum showed intense reactivity with perinuclear vesicles, characteristic of lysosomallocalization. Less intense staining was also observed at the cellsurface in both permeabilized and nonpermeabilized rat2 cells.Lippincott-Schwartz and Fambrough (37) have shown thatthere is a continuous and rapid shuttling of chicken LAMP-1(Lep-100) among the plasma membrane, endosomes, and ly-sosomes in chick embryo fibroblast cells. Cell surface uptake offluorescently labeled monoclonal antibodies, specific for Lep-100, was followed from the endosomes to the lysosomes.LAMP-1 has been detected on the cell surface of the murinemacrophage-like cell line P388, but not NIH 3T3 fibroblastcells (33). This is the first report of cell surface localization ofLAMP-1 on fibroblast cells. LAMP-1 has also been detected atthe surface of human U937 and HL60 myelomonocytic leukemia cell lines but was absent on a variety of other tissue culturecells or normal peripheral blood monocytes (38). When thesecells were induced to differentiate, LAMP-1 was no longerdetectable at the cell surface. These observations suggest that,in addition to the processing of LAMP-1 oligosaccharides, cellsurface expression of the glycoprotein may also be oncodevel-

opmentally regulated. Heuser has recently shown that loweringcytoplasmic pH to 6.5, in a number of cell lines, leads to arapid and reversible movement of lysosomes to the cell periphery (39). This movement follows the distribution of microtu-bules and is associated with decreased membrane ruffling andpseudopodial extensions. Lysosomal proximity and fusion withthe tumor cell surface may be induced by the acidic environmentof anaerobic tumors or other environmental stimuli. A combination of increased levels of P2B/LAMP-1 at the tumor surfaceand expression of more highly branched, sialylated oligosaccharide structures on this glycoprotein may be associated withsecretion of lysosomal hydrolases, decreased cell adhesion toextracellular matrix, and increased cell motility and invasivepotential.

Increased ßl-6branching at the trimannosyl core of complex-type ASM-linked oligosaccharides has previously been associated with transformation of NIH 3T3 cells by N-ras or c-H-ras(40), Rous sarcoma virus-transformed chick embryo fibroblasts(9) and BHK cells (41), and polyoma virus-transformed BHK

cells (8). A comparison of Grade II and Grade III uroepithelialcell lines shows that those cell lines which are tumorigenic invivo and invasive in vitro (grade III) possess more highlybranched oligosaccharides on their cell surface (42). Recently,we have shown that increased ßl-6branching is associated withenhanced metastatic potential in several experimental tumormodels (10). Several observations at the cellular level havesuggested that loss of sialylated lactosamine in ASM-linkedcomplex-type oligosaccharides of metastatic tumor cells is directly associated with increased adhesion to extracellular matrixproteins. Both the Class 1 somatic mutants and the drugswainsonine, which blocks asparginine-linked oligosaccharideprocessing prior to the initiation of the /31-6-linked antenna,enhance tumor cell adhesion and inhibit metastasis (13, 43). In

addition, branched complex-type oligosaccharides isolated fromMDAY-D2 cells were found to inhibit Class 1 cell adhesion toluminili (44). To determine whether the relevant branchedoligosaccharides were those on P2B/LAMP-1, the purifiedglycoprotein was tested for binding activity on extracellularmatrix proteins. The glycoprotein bound to an RGD (Arg-Gly-Asp) affinity column but poorly to extracellular matrixproteins. However, following the removal of sialic acid, poly-lactosamine, or complete ASM-linked oligosaccharides, purified P2B/LAMP-1 bound to collagen type I, fibronectin, andlaminin (15). Therefore, highly sialylated and polylactosamine-containing P2B/LAMP-1 expressed on the surface of metastatic tumors may contribute to the poorly adhesive and motilephenotype of the cells. The branched oligosaccharides containing polylactosamine sequences and terminal sialylation may beas large as 80 to 100 A (45), spanning more than the predicteddiameter of a P2B/LAMP-1 sized protein if it assumed aglobular configuration. Therefore, in addition to modulatingthe interaction between P2B/LAMP-1 and the extracellularmatrix, the large oligosaccharides on LAMP-1 may also inhibitthe interaction between other adhesion receptors and theirligands. Evidence favoring the latter mechanism has recentlybeen reported for the antiadhesion effects of polysialic acidsequences in asparginine-linked oligosaccharides of the embryonic neural cell adhesion molecule (N-CAM) (46).

ACKNOWLEDGMENTS

The authors would like to thank Dr. Korczack and Dr. Brietman fortheir generous gift of RNA.

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1989;49:6077-6084. Cancer Res   M. Heffernan, S. Yousefi and J. W. Dennis  Target of a Metastasis-associated Oligosaccharide StructureMolecular Characterization of P2B/LAMP-1, a Major Protein

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