The disulfide structure of mouse lysosome-associated membrane protein 1.

The disulfide structure of mouse lysosome-associated membrane protein 1 has been determined by reverse-phase isolation and sequence analysis of the cysteine-containing tryptic fragments of the reduced and non-reduced deglycosylated protein. Half-cystines were distinguished (a) by their localization within tryptic or chymotryptic peptides that formed reverse-phase peaks unique to the reduced digests and (b) by their 3H-carboxymethylation only after reduction of the protein. The disulfide arrangement of the cysteines was assigned after isolation of disulfide-linked peptide pairs. Each pair chromatographed as a peak present in the nonreduced (but not the corresponding reduced) tryptic digest. NH2-terminal sequencing as well as reduction, alkylation, and rechromatography of the disulfide-linked fragments led to the following assignment of disulfide bonds: Cys11 and Cys50, Cys125 and Cys161, Cys198 and Cys235, and Cys303 and Cys340. This structure creates four 36-38-residue loops that are symmetrically placed within the two halves of the protein's intraluminal domain. The loops formed by the Cys11-Cys50 and Cys198-Cys235 bridges are homologous, and the Cys125-Cys161 and Cys303-cys340 loops form a second set of homologous domains. The conservation of cysteine residues among lysosome-associated membrane proteins 1 and 2 suggests that this disulfide arrangement is common to both members of this family of lysosomal membrane glycoproteins.

The conservation of cysteine residues among lysosome-associated membrane proteins 1 and 2 suggests that this disulfide arrangement is common to both members of this family of lysosomal membrane glycoproteins.
mLAMP-1 1 (1,2) is a member of a highly conserved family of lysosomal membrane glycoproteins that also includes human LAMP-l (3,4), chicken LEPlOO (5), and rat lgp120 (6) (reviewed in Ref. 7). These proteins are major components of the lysosomal membrane, and several of them have also been found on the plasma membrane of some embryonic, transformed, and metastatic cell types (3,8,9). In addition to a presumed role in the lysosome, these glycoproteins may function as regulated receptors for the extracellular matrix and play a role in the malignant transformation of cells. increased metastatic potential in mouse lymphoma cells (10). In addition, mLAMP-1 molecules bind to an Arg-Gly-Asp (RGD) peptide column; and the deglycosylated form of the molecule binds to collagen, laminin, and fibronectin (11). Another potential function for LAMP-l has been suggested by Febbraito et al. (12), who recently found that surface expression of human LAMP-l, although low on resting platelets, is markedly increased on thrombin-activated platelets and may function in platelet adhesion.
The primary structure of mLAMP-1, deduced from a cDNA clone (2), consists of a 382-residue (42-kDa) protein with a large (346-residue) extracytoplasmic NH*-terminal domain followed by a 24-residue hydrophobic transmembrane region and a short (12-residue) COOH-terminal cytoplasmic tail. The extracytoplasmic domain of mLAMP-1 is composed of two -160-residue homology units that are separated by a Pro/ Ser-rich region. Each of these homologous domains contains 4 uniformly spaced cysteine residues, with two intercysteine intervals of 36-38 residues and one of 68 or 76 residues. The LAMP-l proteins from each of the animal species contain these 8 regularly spaced cysteines as well as many of the same flanking residues. Moreover, a second class of lysosomal membrane glycoproteins, the LAMP-2 family, which have about 30% sequence homology to the LAMP-l molecules, also contains these 8 regularly spaced cysteines and several common surrounding residues (6,13).* The regular spacing of these residues and the presence of some sequences characteristic of immunoglobulins also suggested the possible occurrence of a pair of disulfide-bridged immunoglobulin-like domains (2,13). In this study, the disulfide structure of mLAMP-1 has been elucidated. The data provide evidence that the first and second, third and fourth, fifth and sixth, and seventh and eighth cysteines are disulfide-linked in this protein.
EXPERIMENTAL PROCEDURES AND RESULTS3

DISCUSSION
The data presented in this study collectively indicate that the 8 Cys residues in mLAMP-1 form four intrachain disulfide bonds. Each bond links a pair of adjacent Cys residues such that Cys" and Cys"', Cyslz5 and Cys"l, CYS'~' and Cysz3", and Cys"'" and CYSTS" are bridged within the molecule. These bridges create four 36-38-residue loops in the protein (Fig. 6 There is no homology between the disulfide-bonded domains within the same homology unit. However, the disulfide-linked domains between the two units are homologous. Hence, the loop formed by Cys'l and Cys5" in the first unit is homologous to the Cy~'~'-Cys 235 loop in the second unit (25% identical and 48% similar residues), and the domain formed by Cys?' and Cys" in the first unit is homologous to the Cys303-Cys340 loop in the second unit (21% identical and 48% similar residues) (Fig. 7). 6 The mLAMP-1 molecule contains 20 Asn-X-Ser/Thr glycosylation sites in its extracytoplasmic domain, most of which appear to be modified with complex N-linked sugar moieties (28). The loops formed by the disulfide bonds between Cysli and Cys", Cys"' and Cys16', and Cys"' and CYS'~~ each contain several potential N-linked oligosaccharide sites, whereas the fourth loop, formed by Cys303 and CYSTS', has none. This study revealed that one of those sites, Asnzz4 on the Cy~"~-Cys 235 loop, was not glycosylated since there was an 87-100% stepwise yield of the nonglycosylatedphenylthiohydantoin-Asn during sequencing of the Cys235-containing peptide (Tables I and III). If the residue had been glycosylated, the terminal GlcNAc would have remained attached to the Asn after deglycosylation with trifluoromethanesulfonic acid (17). Therefore, the molecule probably contains 19 N-linked oligosaccharides.
Asn', Asn46, Asn'35, Asnlgo, Asnlg5, Asnzz8, and Asnz7' were found to be actual sites for attachment of Nlinked oligosaccharides since a phenylthiohydantoin-Asn was not recovered at the appropriate site, and in several of these 6 Percentage identical residues was assigned by the SEQHP program (GenMenu), and the percentage conserved residues was calculated by the ALIGN program (GenMenu).
cases, an actual phenylthiohydantoin-Asn-GlcNAc was sequenced at that residue (Tables I and III).
The loops created by the disulfide bonds would not necessarily cause the protein to fold into a highly globular structure since the bridging of neighboring Cys residues creates relatively small loops. Hence, no major change in mobility on sodium dodecyl sulfate-polyacrylamide electrophoresis gel was observed between the reduced and nonreduced species. However, the disulfide bonds apparently stabilize the protein structure, as evidenced by the inability of several proteases to cleave the nonreduced protein under strongly denaturing conditions. The carbohydrate also protected the molecule from proteolytic digestion. The disulfide bonds and abundant oligosaccharides may thus protect the protein against protease digestion in the hostile lysosomal environment (29). The  program of secondary structures predicts that each of the loops formed by the disulfide bonds contains P-sheets. The disulfide bonds may therefore stabilize the tertiary sheet structure.
The importance of the Cys residues to the molecule is further suggested by the complete conservation of the cysteines between the LAMP-l and LAMP-2 groups of proteins. Since the gross structures of these proteins are very similar, they may all have similar disulfide bonding arrangements. Presumably, the role of the disulfide bond is of great enough importance to the structure and/or function of these molecules that these bonds have been conserved throughout evolution of the various animal species (human, mouse, rat, and chicken) as well as between the two different groups of molecules (LAMP-l and LAMP-2). The regular spacing of the Cys residues together with the presence of a characteristic immunoglobulin-related sequence common to the V-like domains, Tyr-X-Cys (31), had earlier suggested that the mLAMP-1 molecule might fall into the immunoglobulin superfamily of genes (2,13). The data generated during this study, however, suggest that mLAMP-1 is not a member of this superfamily.
Specifically, the loops formed in the immunoglobulins are composed of -70 residues, whereas the mLAMP-1 loops are of 36-38 residues. This alone would not exclude LAMP-l molecules from membership in the immunoglobulin superfamily because another member of the immunoglobulin family, the Fc receptor, has relatively small loops (18). However, the Tyr-X-Cys sequences found at CyP and Cys303 in mLAMP-1 do not fall at the carboxylterminal end of a disulfide loop in mLAMP-1 as they do in the immunoglobulins.
This observation together with the fact that there are no other sequences characteristic of the immunoglobulins in the mLAMP-1 sequence lead us to conclude that mLAMP-1 is not a member of the immunoglobulin superfamily of genes. During a broad protein search, no proteins outside of the LAMP-l or LAMP-2 family of proteins were found to be homologous to mLAMP-1 or to contain domains homologous to the disulfide-linked loops of this protein. 7