Intracellular assembly of Kell and XK blood group proteins.

Kell, a 93 kDa type II membrane glycoprotein, and XK, a 444 amino acid multi-pass membrane protein, are blood group proteins that exist as a disulfide-bonded complex on human red cells. The mechanism of Kell/XK assembly was studied in transfected COS cells co-expressing Kell and XK proteins. Time course studies combined with endonuclease-H treatment and cell fractionation showed that Kell and XK are assembled in the endoplasmic reticulum. At later times the Kell component of the complex was not cleaved by endonuclease-H, indicating N-linked oligosaccharide processing and transport of the complex to a Golgi and/or a post-Golgi cell fraction. Surface-labeling of transfected COS cells, expressing both Kell and XK, demonstrated that the Kell/XK complex travels to the plasma membrane. XK expressed in the absence of Kell was also transported to the cell surface indicating that linkage of Kell and XK is not obligatory for cell surface expression.     

Expression profiles of mouse Kell, XK, and XPLAC mRNA.

Kell and XK are related because in red cells they exist as a disulfide-bonded complex. Kell is an endothelin-3-converting enzyme, and XK is predicted to be a transporter. Absence of XK, which is accompanied by reduced Kell on red cells, results in acanthocytosis and late-onset forms of central nervous system and neuromuscular abnormalities that characterize the McLeod syndrome. In this study, expression of mouse XK, XPLAC, a homolog of XK, and Kell were compared by in situ hybridization histochemistry (ISHH) and RT-PCR. ISHH showed that Kell and XK are coexpressed in erythroid tissues. ISHH detected XK, but not Kell, mRNA in testis, but RT-PCR indicated that both Kell and XK are coexpressed. XK, but not Kell, was significantly expressed in brain, spinal cord, small intestine, heart, stomach, bladder, and kidney. ISHH did not detect XK in skeletal muscle but RT-PCR did. In brain, XK was predominantly expressed in neuronal rather than in supportive cells. By contrast, XPLAC was predominantly expressed in the thymus. Coexpression of Kell and XK in erythroid tissues and the different expressions in non-erythroid tissues suggest that XK may have a complementary hematological function with Kell and a separate role in other tissues.     

Endothelin-3-converting enzyme activity of the KEL1 and KEL6 phenotypes of the Kell blood group system

The Kell blood group protein is a metalloendopeptidase that preferentially cleaves a Trp(21)-Ile(22) bond of big endothelin-3 producing bioactive endothelin-3. Kell is a polymorphic protein, and 25 different phenotypes, because of point mutations resulting in single amino acid substitutions, have been described. It was recently reported that a recombinant form of KEL1 (K, K1) phenotype, expressed in K562 and HEK293 cells, had no endothelin-3-converting activity, in contrast to the common KEL2 (k, K2) phenotype. We demonstrate that KEL1 red blood cells and also a soluble recombinant form of KEL1 protein (s-Kell KEL1) have similar enzymatic activity as the common Kell phenotype. In addition we show that KEL6 red blood cells, which are more prevalent in persons of African heritage than in Caucasians also have endothelin-3-converting enzyme activity and that the recombinant soluble form of KEL6 protein (s-Kell KEL6) has similar K(m) values as the wild-type.     

Structure and expression of the mouse homologue of the XK gene

 The human Kx blood group antigen is carried by a 37 000 M _r apparent molecular mass membrane polypeptide which is deficient in rare individuals with the McLeod syndrome. The X-linked human XK gene is transcribed in many tissues including adult skeletal muscle and brain, sieges of disorders observed in McLeod syndrome. We report here the cloning of the orthologous mouse XK mRNA. Comparison of XK from human and mouse revealed 80% sequence similarity at the amino acid level. The mouse XK gene is organized in two exons and is expressed in many tissues, but its expression pattern is slightly different from that of the human gene. The presence in mouse erythrocyte membrane of a 43 000 M _r Kx-related protein was demonstrated by immunoblotting with a rabbit antiserum directed against the human protein. With non-reduced samples, a 140 000 M _r species was detected instead of the 43 000 M _r protein, suggesting that, as demonstrated in the Kx polypeptide might be complexed with another protein in mouse red cells, presumably the homologue of the human Kell protein of 93 000 M _r. Received: 22 February 1999 / Revised: 8 June 1999     

Kell blood group antigens are part of a 93,000-dalton red cell membrane protein

Monospecific Kell blood group antibodies, of either human alloimmune or mouse monoclonal origin, react with a single surface-exposed protein of 93,000 daltons. Chymotryptic peptide maps of the 93,000-dalton protein isolated by antibodies of two different specificities (anti-K7 or anti-K14) indicate that Kell epitopes reside on the same protein. Kell protein is similar in size to band 3 protein but differs markedly in its tryptic and chymotryptic peptide maps, indicating that they are different proteins. In addition, sheep antibody to human band 3 does not react with Kell protein. Rabbit antibody to Kell protein reacts, by Western immunoblotting, with membrane proteins from Kell antigen positive red blood cells but not from those of a Ko (Kell null) cell. In intact red cells only a small portion of the Kell protein is available to lactoperoxidase-catalyzed iodination. Under nonreducing conditions Kell antigen is isolated not only as a 93,000-dalton protein but also as larger protein complexes ranging in size from above 200,000 to 115,000 daltons. Treatment of red cells with iodoacetamide, prior to isolation of Kell protein, reduces the amount of the very large complexes, but Kell protein occurs both as 115,000- and 93,000-dalton proteins.     

Purification and characterization of an estrogen-inducible membrane glycoprotein. Evidence that it is a transferrin receptor

A number of N-linked membrane glycoproteins are induced during chick oviduct differentiation. We have purified a major estrogen-inducible glycoprotein (Mr = 91,000) to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of partial NH2-terminal sequence data with membrane glycoproteins having similar Mr showed a limited homology with human and murine transferrin receptors. We observed that oviduct membranes contain estrogen-inducible transferrin receptor activity (Kd = 2-8 x 10(-8) M). Analytical purification of the putative receptor on an ovotransferrin-Affi-Gel affinity column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis reveals a protein of Mr, 180,000, which contains two disulfide-linked subunits of Mr 91,000. The receptor reacts very strongly with antibodies prepared against the 91-kDa glycoprotein on Western blots. Western blot analysis confirms that the 91-kDa glycoprotein is induced by estrogen. The protein has 2% total carbohydrate with Man, GlcNAc, Gal, GalNAc, and NeuAc in a molar ratio of 6:4:2:1:1. The protein contains at least one O-linked moiety. Analysis of the O-linked moiety by glycosidase digestions and gel filtration indicates there are sialo tetra- and trisaccharides and a neutral disaccharide(s). Labeled N-linked glycopeptides were prepared by pronase digestion, beta-elimination, and 3H-acetylation. The N-linked oligosaccharides include high mannose and complex neutral nonbisected biantennary types in an approximate ratio of 3:1 as determined by serial lectin affinity chromatography.     

Molecular cloning of gp49, a cell-surface antigen that is preferentially expressed by mouse mast cell progenitors and is a new member of the immunoglobulin superfamily.

gp49 is a Mr 49,000 glycoprotein expressed on the surface of mouse bone marrow-derived mast cells, which are progenitors for the major in vivo mast cell subclasses, typified by intestinal mucosal mast cells and serosal mast cells. The amino-terminal amino acid sequence of gp49 was determined after isolation of the solubilized membrane protein by affinity chromatography with the B23.1 anti-gp49 monoclonal antibody. Redundant oligonucleotides were used to isolate a full-length 1.3-kilobase cDNA from a mouse mast cell library. The predicted amino acid sequence contains a signal peptide of 23 residues, an extracellular domain of 215 residues with three potential sites of N-linked glycosylation, a transmembrane domain of 23 residues, and a cytoplasmic tail of 42 residues. Hybridization of the gp49 cDNA was limited to mRNA extracted from those cell types that also bound the B23.1 monoclonal antibody as assessed by cytofluorographic analyses. The predicted extracellular domain of gp49 contains two regions of 48 and 51 amino acids, each flanked by cysteine residues. Both regions meet criteria for being C2-type domains of the immunoglobulin superfamily based upon the alignment of consensus amino acids and their predicted secondary structure organization. Thus, gp49, a membrane glycoprotein preferentially expressed by the progenitor mast cell population, is a new member of the immunoglobulin superfamily.     

The murine homologue of the T lymphocyte antigen CD28. Molecular cloning and cell surface expression

The human T lymphocyte Ag CD28 (Tp44) is a homodimeric glycoprotein expressed on the surface of a majority of human peripheral T cells and thymocytes. Although exposure of T cells to anti-CD28 mAb does not activate T cells, stimulation of CD28 can synergize with signals transmitted through the TCR or other stimuli to augment proliferation and lymphokine production. We have used a portion of the human CD28 cDNA to isolate a homologous murine cDNA from an EL4 T lymphoma library. The murine clone has 61% nucleotide identity with the human cDNA. Both human and murine sequences exhibit homology with members of the Ig supergene family and CTLA-4, a T cell specific murine gene. Many characteristics of the human CD28 molecule are conserved within the putative murine CD28 polypeptide. The murine cDNA sequence encodes a polypeptide of 218 amino acids that has 68% identity with the human sequence. Both the murine and human molecules are integral membrane glycoproteins with hydrophobic signal peptide sequences and transmembrane region. All five potential N-linked glycosylation sites are conserved and six of the seven cysteine residues of the mouse protein are found in the human CD28 polypeptide. The murine cDNA is encoded by a single copy nonrearranging gene whose expression at the mRNA level is restricted to T cells. A rabbit antiserum was raised against a synthetic peptide corresponding to a hydrophilic portion of the translated murine cDNA sequence. This antiserum identifies an 80-kDa homodimer consisting of disulfide-bonded subunits of 40 kDa that is expressed on splenic T cells, thymocytes, and several T cell tumors, but not on B cells. deglycosylation studies indicate that four of the five N-linked glycosylation sites are used and that the mature core protein has a molecular mass of 25 kDa, close to that predicted by the cDNA sequence. Transfection of the murine cDNA into Chinese hamster ovary cells resulted in the expression of an 80-kDa dimeric molecule that was immunoprecipitated by the antipeptide antiserum. Taken together, these data provide strong support that we have identified the murine homologue of CD28.     

Kx,a Quantitatively Minor Protein from Human Erythrocytes,Is Palmitoylatedin Vivo

Kx is a quantitatively minor blood group protein of human erythrocytes which is thought to be a membrane transporter. In the red cell membrane, Kx forms a complex stabilized by a disulfide bond with the Kell blood group membrane protein which might function as a metalloprotease. The palmitoylation status of these proteins was studied by incubating red cells with [³H] palmitic acid. Purification of the Kell-Kx complex, by immunochromatography on an immobilized human monoclonal antibody of Kell blood group specificity demonstrated that the Kx but not the Kell protein is palmitoylated. Six cysteines in Kx are predicted to be intracytoplasmic and might be targets for palmitoylation. Three of these cysteines are present in a portion of sequence which is predicted to form an amphipathic alpha helix. Palmitoylation of one or several of these cysteines might contribute to anchor the cytoplasmic portion of the Kx protein to the inner surface of red cell membrane.     

Molecular cloning, expression, and chromosomal localization of a human tubulointerstitial nephritis antigen

Tubulointerstitial nephritis antigen (TIN-ag) is an extracellular matrix basement protein which was originally identified as a target antigen involved in anti-tubular basement membrane (TBM) antibody-mediated interstitial nephritis (TIN). Further investigations elucidated that TIN-ag plays a role in renal tubulogenesis and that TIN-ag is defected in hereditary tubulointerstitial disorder such as juvenile nephronophthisis. We previously isolated and characterized 54 kDa glycoprotein as TIN-ag. cDNA encoding rabbit and mouse TIN-ag has recently been identified. In the present study, the cDNA of the human homologue of TIN-ag was cloned and its nucleotide sequence was determined (Accession No. AB022277; the DDBJ nucleotide sequence database). Deduced amino acid sequence (476 aa) exhibited the presence of a signal peptide (1-18 aa), cysteine residues termed follistatin module, six potential glycosylation sites, and an ATP/GTP-binding site. Homology search revealed approximately 85% homology with both rabbit and mouse TIN-ag, and also some ( approximately 40%) similarity with C. elegans. Human TIN-ag contained a sequence similar to several classes of extracellular matrix molecules in amino terminal region and to cathepsin family of cysteine proteinases in the carboxyl terminal region. Northern blot analysis revealed exclusive expression of this molecule in human adult and fetal kidney tissues. Using a monoclonal antibody recognizing human TIN-ag, protein expression ( approximately 50 kDa) was identified in cultured COS-1 cells transfected with human TIN-ag cDNA. The human TIN-ag was mapped to chromosome 6p11.2-12 by fluorescence in situ hybridization. These results may provide further evidence for understanding TIN-ag molecule and clues for gene analysis of juvenile nephronophthisis.     

Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis.

The surface receptor for urokinase plasminogen activator (uPAR) has been recognized in recent years as a key molecule in regulating plasminogen mediated extracellular proteolysis. Surface plasminogen activation controls the connections between cells, basement membrane and extracellular matrix, and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human fibroblasts [Okayama and Berg (1983) Mol. Cell Biol., 3, 280-289]. The cDNA encodes a protein of 313 amino acids, preceded by a 21 residue signal peptide. A hydrophobicity plot suggests the presence of a membrane spanning domain close to the C-terminus. The cDNA hybridizes to a 1.4 kb mRNA from human cells, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis, direct binding studies and cross-linking experiments show that the transfected mouse LB6 cells specifically bind human uPA, which in turn activates plasminogen. The Mr of the mature human receptor expressed in mouse cells is approximately 55,000, in accordance with the naturally occurring, highly glycosylated human uPAR. The Mr calculated on the basis of the cDNA sequence, approximately 35,000, agrees well with that of the deglycosylated receptor.     

Identification of human galactoprotein b3, an oncogenic transformation-induced membrane glycoprotein, as VLA-3 alpha subunit: the primary structure of human integrin alpha 3.

Galactoprotein b3 is one of the cell membrane glycoproteins of fibroblasts showing enhanced expression in association with oncogenic transformation. Analysis of cDNA for this glycoprotein from hamster fibroblasts indicated that the glycoprotein is a member of the integrin superfamily [Tsuji, T., Yamamoto, F., Miura, Y., Takio, K., Titani, K., Pawar, S., Osawa, T., & hakomori, S. (1990) J. Biol. Chem. 265, 7016-7021]. In the present study, we examined the change in the amounts of mRNA for the mouse and human counterparts in fibroblasts after oncogenic transformation by Northern blot analysis using hamster galactoprotein b3 cDNA as a probe. In both human and murine fibroblasts transformed with SV-40, the homologous mRNA to galactoprotein b3 was also found to increase as compared with the progenitor cells. The human homologue of galactoprotein b3 cDNA was cloned from human bladder carcinoma cell line (T24) cDNA library. The cDNA codes for a single polypeptide of which the N-terminal sequence (21 amino acids) is identical with that of human VLA-3 alpha subunit. Based on this sequence identity and the structural similarities (i.e. the positions of most cysteine residues, the presence of a transmembrane domain near the C-terminus and the presence of metal binding sequences) with other integrins so far cloned, we conclude that human galactoprotein b3 is an integrin alpha 3 subunit. The mature integrin alpha 3 polypeptide was composed of 1,019 amino acid residues, and the overall structure was quite similar to the hamster counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a cDNA that encodes the peptide core of the secretory granule proteoglycan of human promyelocytic leukemia HL-60 cells

A cDNA that encodes the peptide core of the secretory granule proteoglycan of the human promyelocytic leukemic cell line, HL-60, has been isolated and analyzed. When human genomic DNA was digested and probed under conditions of low stringency with a rat cDNA that encodes a Mr = 18,600 serine/glycine-rich proteoglycan peptide core in L2 yolk sac tumor cells (Bourdon, M. A., Oldberg, A., Pierschbacher, M., and Ruoslahti, E. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1321-1325) and basophilic leukemia-1 cells (Avraham, S., Stevens, R. L., Gartner, M. C., Austen, K. F., Lalley, P. A., and Weis, J. H. (1988) J. Biol. Chem. 263, 7292-7296), a number of DNA fragments were identified. A HL-60 cell-derived cDNA library was therefore screened under conditions of low stringency with the rat probe to identify and isolate a human homologue of this rat proteoglycan peptide core. Analysis of the resulting human cDNA clones indicated that the proteoglycan peptide core that is expressed in HL-60 cells is Mr = 17,600 and contains an 18-amino acid glycosaminoglycan attachment region that consists primarily of alternating serin and glycine. Northern blot analysis of total RNA probed with the human cDNA revealed that the major message for this proteoglycan peptide core in HL-60 cells is approximately 1.3 kilobase pairs in size. When a Southern blot of digested human genomic DNA was probed with the human cDNA, three bands of approximately 6, 9, and 12 kilobase pairs were detected. However, when the Southern blot was probed with the XmnI----3' fragment of this human cDNA, one prominent band was detected, indicating that a single gene encodes this protein in the human. Analysis of the DNA from human/mouse and human/hamster somatic cell hybrids probed with the human cDNA demonstrated that the gene that encodes this molecule resides on human chromosome 10. Because the proteoglycans that are present in the secretory granules of different types of rat and mouse mast cells possess small peptide cores that are rich in serine and glycine, we propose that this HL-60 cell-3 derived cDNA encodes the peptide core of the proteoglycan that is expressed in the secretory granules of this human promyelocytic cell.     

Molecular cloning of the murine homologue of CD2. Homology of the molecule to its human counterpart T11

In order to characterize the mouse homologue of the CD2 molecule, which has not yet been identified by immunologic means, cDNA clones putatively encoding mouse CD2 have been isolated by cross-hybridization with a cDNA probe for rat CD2. The predicted amino acid sequence of the putative mouse CD2 protein is consistent with that of a transmembrane glycoprotein, i.e., it consists of an N-terminal region of 186 amino acids bearing six potential N-glycosylation sites, a hydrophobic transmembrane segment of 25 residues, and a large cytoplasmic region of 116 amino acids rich in proline and basic residues. Comparison with the human and rat CD2 sequences clearly indicated the predicted mouse protein to be the mouse equivalent. Striking evolutionary conservations between mouse, rat, and human CD2 were found in their cytoplasmic region, suggesting a functional consequence of that segment for the physiologic role of CD2, such as signal transduction. RNA blot hybridization analysis demonstrated the expression of CD2 in T lymphocytes and in the NK cell lineage in mice. These data strongly suggest that the putative mouse CD2 molecule may perform some biologic functions in mouse T lymphocytes and NK cells as documented in humans.     

Cell surface glycoproteins involved in the stimulation of interleukin 1-dependent interleukin 2 production by a subline of EL4 thymoma cells. II. Structure, biosynthesis, and maturation

In the present study, we examined the biosynthesis and the maturation of two distinct membrane glycoproteins detected by two monoclonal antibodies (RL388 and RL119), which were selected on the basis of their ability to stimulate the production of interleukin 2 by a subline of the murine EL4 thymoma. RL388 detected a disulfide-linked heterodimer complex (Mr = 130,000) composed of a glycosylated heavy (Mr = 86,000) and a nonglycosylated light (Mr = 39,000) subunit. The unglycosylated precursor of the heavy chain was a polypeptide of Mr = 57,500, which was converted upon maturation into a Mr = 73,000 core-glycosylated intermediate, and then into the Mr = 86,000 surface-expressed molecule. Partial endo-H digestion of the core-glycosylated form suggested the presence of four N-linked glycan units. The antibody reacted with a protein determinant expressed on the mature form as well as the unglycosylated precursor of the heavy chain. Moreover, both subunits assembled rapidly during biosynthesis, and the glycosylation of the heavy chain was not required for this association. Taken together, these data suggest that the antigen detected by RL388 may be the murine homologue of the human 4F2 antigen. The antigen identified by RL119 was a surface glycoprotein of Mr = 55,000 with three to five N-linked glycan units. The unglycosylated precursor polypeptide was of Mr = 29,000. The fully core-glycosylated form of Mr = 41,000, which was detected after inhibition of glucosidase I with 1-deoxynojirimycin, was converted into a Mr = 39,000 intermediate, and upon further trimming, into a Mr = 36,000 endo-H-sensitive form. The latter could be detected for chase periods of over several hours, thus suggesting a low rate of intracellular processing. The wide cellular distribution of the molecules identified by RL388 and RL119 and their preferential expression on the surface of growing cells suggests that they may be associated with cell activation events.     

Molecular cloning and characterization of GalNAc 4-sulfotransferase expressed in human pituitary gland

We have previously cloned chondroitin-4-sulfotransferase (C4ST) cDNA from mouse brain. In this paper, we report cloning and characterization of GalNAc 4-sulfotransferase (GalNAc4ST), which transfers sulfate to position 4 of the nonreducing terminal GalNAc residue. The obtained cDNA contains a single open reading frame that predicts a type II transmembrane protein composed of 424 amino acid residues. Identity of the amino acid sequence between GalNAc4ST and human C4ST was 30%. When the cDNA was transfected in COS-7 cells, sulfotransferase activity toward carbonic anhydrase VI was overexpressed but no sulfotransferase activity toward chondroitin or desulfated dermatan sulfate was increased over the control. Sulfation of carbonic anhydrase VI by the recombinant GalNAc4ST occurred at position 4 of the GalNAc residue of N-linked oligosaccharides. The recombinant GalNAc4ST transferred sulfate to position 4 of GalNAc residue of p-nitrophenyl GalNAc, indicating that this sulfotransferase transfers sulfate to position 4 at the nonreducing terminal GalNAc residue. Dot blot analysis showed that the message of GalNAc4ST was expressed strongly in the human pituitary, suggesting that the cloned GalNAc4ST may be involved in the synthesis of the nonreducing terminal GalNAc 4-sulfate residues found in the N-linked oligosaccharides of pituitary glycoprotein hormones.     

MDC9, a widely expressed cellular disintegrin containing cytoplasmic SH3 ligand domains

Cellular disintegrins are a family of proteins that are related to snake venom integrin ligands and metalloproteases. We have cloned and sequenced the mouse and human homologue of a widely expressed cellular disintegrin, which we have termed MDC9 (for metalloprotease/disintegrin/cysteine-rich protein 9). The deduced mouse and human protein sequences are 82% identical. MDC9 contains several distinct protein domains: a signal sequence is followed by a prodomain and a domain with sequence similarity to snake venom metalloproteases, a disintegrin domain, a cysteine-rich region, an EGF repeat, a membrane anchor, and a cytoplasmic tail. The cytoplasmic tail of MDC9 has two proline-rich sequences which can bind the SH3 domain of Src, and may therefore function as SH3 ligand domains. Western blot analysis shows that MDC9 is an approximately 84-kD glycoprotein in all mouse tissues examined, and in NIH 3T3 fibroblast and C2C12 myoblast mouse cell lines. MDC9 can be both cell surface biotinylated and 125I-labeled in NIH 3T3 mouse fibroblasts, indicating that the protein is present on the plasma membrane. Expression of MDC9 in COS-7 cells yields an 84-kD protein, and immunofluorescence analysis of COS-7 cells expressing MDC9 shows a staining pattern that is consistent with a plasma membrane localization. The apparent molecular mass of 84 kD suggests that MDC9 contains a membrane-anchored metalloprotease and disintegrin domain. We propose that MDC9 might function as a membrane-anchored integrin ligand or metalloprotease, or that MDC9 may combine both activities in one protein.     

The cDNA sequence of mouse LAMP-2. Evidence for two classes of lysosomal membrane glycoproteins

We describe the isolation and sequencing of a cDNA encoding the mouse lysosomal membrane glycoprotein mLAMP-2 and the sequence differences that distinguish this molecule from the LAMP-1 class of proteins. An oligonucleotide probe corresponding to the NH2-terminal amino acid sequence of purified mLAMP-2 was synthesized by the polymerase chain reaction and used to screen several cDNA libraries. cDNA clones with an insert of 1,700 nucleotides were identified and sequenced. The deduced amino acid sequence of mLAMP-2 comprises a signal sequence of 25 residues and a 390-amino acid polypeptide (Mr 43,017) with the following putative domains: a large intraluminal region (residues 1-354) with 17 N-linked glycosylation sites (Asn-X-Ser/Thr), a hydrophobic transmembrane-spanning region of 24 residues (355-378), and a COOH-terminal cytoplasmic tail of 12 residues (379-390). When this sequence is compared with those of other lysosomal membrane glycoproteins, it is apparent that mouse LAMP-2 and human LAMP-2 form one homology class (LAMP-2) that is separated from the LAMP-1 class of proteins. The sequence differences in these two classes provide a basis for comparing the structure of the proteins with their biochemical and biological properties.