| Budget Amount *help |
¥5,700,000 (Direct Cost: ¥5,700,000)
Fiscal Year 1988: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1987: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1986: ¥4,200,000 (Direct Cost: ¥4,200,000)
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| Research Abstract |
The function and structure of the liver mannan-binding protein (MBP), a hepatic lectin specific for mannose and N-acetylglucosamine, were investigated by utilizing gene recombinant technology. The major findings obtained in this sutdy are as follows.
1. The primary structure of rat liver MBP was deduced from its cDNA sequence. cDNA clones encoding rat liver MBP were isolated from a rat liver cDNA library carried in gt 11 by screening with an affinity purified anti-rat liver MBP antibody. The nucleotide sequence of the cDNA determined by the dideoxymethod revealed the complete amino acid sequence of the MBP (226 residues). The NH_2-terminal residue of the MBP, glutamic acid, was preceded by a signal peptide consisting of hydrophobic strecth of 18 amino acids. Near the NH_2-terminal, there was a collagen-like domain, which consisted of 19 repeats of the sequence, Giy-X-Y. The COOH-terminal half of the molecule obtained by collagenase digestion retained the sugar binding activity, revealing that the carbohydrate recognizing domain is located in its COOH terminal 150 amino acid residues. Treatment with reducing agents inactivated the lectin but this effect was reversed by air oxidation, indicating that disulfide bonds are associated with the expression of the binding activity.
2. Identification of endogenous liginds of the liver MBP. Endogenous ligands for the hepatic lectin were isolated from rat liver rough microsomes and primary cultured hepatocytes by affinity chromatography on an immobilized MBP column. Western blotting using specific antisera revealed that biosynthetic intermediates of serum glycoproteins, alpha_1-acid glycoprotein, alpha_1-macroglobulin, and alpha_1-antitrypsin, and a lysosomal enzyme,beta-glucuronidase were the major constituents of the endogenous ligands. These results suggest that the liver MBP is associated with the transport of glycoproteins within the cells during the process of their biosynthesis.
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