MOLECULAR CHARACTERIZATION OF P2B/LAMP-1, A MAJOR PROTEIN TARGET OF A METASTASIS-ASSOCIATED OLIGOSACCHARIDE STRUCTURE

  • 1 November 1989
    • journal article
    • research article
    • Vol. 49  (21) , 6077-6084
Abstract
Sialylated and GlcNAc.beta.1-6Man.alpha.1-6Man.beta.1 (.beta.1-6 branched) complex-type oligosaccharides linked to asparagine residues of membrane glycoproteins in metastatic murine tumor cells have been associated with efficient tumor cell metastasis. A large proportion of these oligosaccharide structures, in several unrelated malignant cell lines, have been shown to be associated with a glycoprotein termed P2B, with a molecular weight of 130,000. This glycoprotein has recently been purified from the metastatic MDAY-D2 cell line and shown to be biochemically similar to a lysosomal associated membrane glycoprotein (LAMP-1). We report here the details of 2147 nucleotide complementary DNA encoding the entire murine P2B polypeptide which was immunoselected from a .lambda.gt11 expression library and sequenced. The sequence is similar to a complementary DNA coding for mouse LAMP-1 with the exception of a 5'' untranslated region, leader signal-sequence, and various insertions, deletions, and substitutions in the 3'' untranslated domain. An open reading frame of 405 amino acids encodes a mature polypeptide of 382 residues with a predicted molecular weight of 42,000. P2B/LAMP-1 possesses 20 asparagine-linked glycosylation sites separated into equal halves by a central, putative hinge region and is anchored by a carboxy, membrane-spanning, domain. Topological considerations dictate that cell surface expression of P2B/LAMP-1 exposes the bulk of the glycoprotein into the extracellular compartment. Immunofluorescent staining of fibroblast cells indicated tha P2B/LAMP-1 was associated with lysosomal membranes and, to a lesser degree, select surfaces of plasma membrane. An amino acid comparison of the murine sequence with its recently cloned rat, human, and chicken counterparts shows a conservation of 17 of 20 asparagine-linked glycosylation consensus sites, eight of eight cysteine residues, and other selected protein domains. The interspecies conservation of these domains suggests that they are important for the structure and function of the P2B/LAMP-1 glycoprotein. Northern analysis revealed that P2B/LAMP-1 is widely expressed in normal murine tissues and tumor cell lines. However, in two experimental models of metastasis, where changes in branching of oligosaccharides on P2B/LAMP-1 have been shown to occur, comparable levels of P2B/LAMP-1 mRNA were found in both metastatic and nonmetastatic cell lines.