Functional expression of human mannan-binding proteins (MBPs) in human hepatoma cell lines infected by recombinant vaccinia virus: post-translational modification, molecular assembly, and differentiation of serum and liver MBP.
Human mannan-binding proteins (MBPs) occur in two forms, serum MBP (S-MBP) and liver MBP (L-MBP), both of which are synthesized in the liver from a single form of human MBP mRNA. To investigate further the mechanisms of post-translational modification, molecular assembly and differentiation of S-MBP and L-MBP in vitro, we expressed a full-length human MBP cDNA in three human hepatoma cell lines, using the vaccinia virus expression system. The expression of human MBP cDNA reproduced the native MBP differentiation of S-MBP and L-MBP in human hepatoma cells. The recombinant S-MBP was secreted into the medium, and the recombinant L-MBP retained in the cells. The former had the ability to activate the complement through the classical or lectin pathway but the latter did not. Furthermore, one notable difference between the two MBPs was the degree of oligomerization through interchain disulfide bonds between subunits. In addition, we showed that both S-MBP and L-MBP undergo hydroxylation of lysine and proline residues in collagen-like sequences, and that the hydroxylysine is glycosylated to form glucosylgalactosylhydroxylysine (GluGalHyl) and galactosylhydroxylysine (GalHyl). Hydroxylation was required for S-MBP to be assembled into large complexes, the apparent molecular sizes of which were estimated to be 200-1,300 kDa by SDS-PAGE under non-reducing conditions and gel filtration under non-denaturing conditions. The hydroxylation and subsequent glycosylation and oligomerization were inhibited by alpha,alpha'-dipyridyl, an inhibitor of collagen lysyl and prolyl hydroxylases. These results suggested that newly synthesized lectins undergo post-translational modifications unique to the two forms of MBP, S-MBP, and L-MBP, in human hepatocytes and hepatoma cells, and that the collagen-like domains of the MBPs play an important role in promoting molecular assembly.
Ma
Y
Y
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University.
Shida
H
H
Kawasaki
T
T
D000368:Aged; D000595:Amino Acid Sequence; D000818:Animals; D001704:Biopolymers; D019556:COS Cells; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000821:virology; D002352:Carrier Proteins / Q000097:blood / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism; D037222:Collectins; D003167:Complement Activation; D006801:Humans; D006900:Hydroxylation; D008099:Liver / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D008297:Male; D008351:Mannans / Q000378:metabolism*; D008969:Molecular Sequence Data; D011392:Proline / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D011995:Recombination, Genetic; D014407:Tumor Cells, Cultured; D014616:Vaccinia virus / Q000235:genetics*
D001704:Biopolymers; D002352:Carrier Proteins; D037222:Collectins; D008351:Mannans; D003094:Collagen; D011392:Proline; D008239:Lysine