|Budget Amount *help
¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1991 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1990 : ¥1,400,000 (Direct Cost : ¥1,400,000)
We used the oligonucleotide probe corresponding to the internal amino acid sequence of a lysosomal membrane glycoprotein with a molecular weight of 85 K (LGP85) and isolated and characterized cDNA clones containing the entire coding region. The isolated cDNA comprised 2665 nucleotides. The predicted amino acid sequences of LGP85 consisted of 478 amino acid residues (Mr. 54, 090) and the protein has 11 potential N-glycosylation sites. Since the NH_2 terminal sequence determined from purified LGP85 was identical to the NH_2 terminal sequence deduced from the nucleotide sequence of the cDNA, except for the lack of initiator methionine which is likely to be cleaved off posttranslationally, it is likely that LGP85 has an uncleavable signal peptide at the NH_2 terminus. Hydropathy plots show that LGP85 possesses two strong hydrophobic regions at the NH_2 terminus (residues 426) and near the COOH terminus (residues 433-457), respectively. Either one or both of the domains might be used for me
mbrane anchoring. A comparison of the sequences of the other lysosomal membrane glycoproteins with that of LGP85 revealed no homology. Glycine-tyrosine residues (so-called GY motif) which are thought an important signal for delivery of lysosomal membrane glycoproteins to lysosomes were not contained in the cytoplasmic tail of LGP85 (residues 458-478). LGP85 appears to be an unique lysosomal membrane glycoprotein that does not require tyrosine residues for targeting to lysosomes. Tyrosine residue may not be an essential signal for delivering newly synthesized lysosomal membrane glycoproteins to lysosomes.
A full length cDNA for a human lysosomal membrane sialoglycoprotein (hLGP85) was isolated as a probe of the cDNA of rat LGP85 (rLGP85) from the cDNA library prepared from total mRNA of QGP-1NL cells, a human pancreatic islet tumor cell with a high metastatic activity. The deduced amino acid sequence shows that hLGP85 consists 478 amino acid residues (MW. 54, 289). The protein has 10 putative N-glycosylation sites and 2 hydrophobic regions at the NH2- and near the COOH-termini, respectively. Thus, both domains probably constitute putative transmembrane domains. It exhibits 86% and 79% sequence similarities in amino acids and nucleic acids to rat lysosomal membrane sialoglycoprotein (rLGP85), respectively. The protein also contained the short cytoplasmic tail at the COOH-terminus which does not form the glycine-tyrosine sequence (GY-motif), socalled lysosomal targetting signal.
We isolated cDNA for LGP107. Mutant LGP107 (M-LGP85) was constructed by deleting the cytoplasmic tail of LGP107. The cDNAs of LGP107 and M-LGP107 were separately transfected to COS cells and intracellular movement of the expressed proteins in the cells were examined with immune-electron microscope. The expressed LGP107 protein was firstly transported to plasma membranes via the Golgi complex and from which the protein moved to lysosomes through endosomes. Although M-LGP107 protein expressed in COS cells took the same route until endosomes as LGP107 protein, the movement from endosomes to lysosomes was completely blocked. The result revealed that the cytoplasmic tail of LGP107 is essential for LGP107 to translocate to lysosomes. Less