Ma Y, Shida H, Kawasaki T
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University.
J Biochem. 1997 Oct;122(4):810-8. doi: 10.1093/oxfordjournals.jbchem.a021827.
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.
人甘露聚糖结合蛋白（MBP）有两种形式，即血清MBP（S-MBP）和肝脏MBP（L-MBP），二者均由肝脏中单一形式的人MBP mRNA合成。为了进一步研究S-MBP和L-MBP在体外的翻译后修饰、分子组装及分化机制，我们利用痘苗病毒表达系统在三种人肝癌细胞系中表达了全长人MBP cDNA。人MBP cDNA的表达重现了人肝癌细胞中S-MBP和L-MBP的天然分化。重组S-MBP分泌到培养基中，而重组L-MBP保留在细胞内。前者有能力通过经典途径或凝集素途径激活补体，而后者则不能。此外，两种MBP之间一个显著的差异是亚基间通过链间二硫键形成的寡聚化程度。另外，我们发现S-MBP和L-MBP在胶原样序列中的赖氨酸和脯氨酸残基均发生羟基化，且羟赖氨酸被糖基化形成葡糖基半乳糖基羟赖氨酸（GluGalHyl）和半乳糖基羟赖氨酸（GalHyl）。羟基化是S-MBP组装成大复合物所必需的，在非还原条件下通过SDS-PAGE以及在非变性条件下通过凝胶过滤估计其表观分子大小为200 - 1300 kDa。胶原赖氨酰和脯氨酰羟化酶的抑制剂α,α'-联吡啶抑制了羟基化以及随后的糖基化和寡聚化。这些结果表明，新合成的凝集素在人肝细胞和肝癌细胞中经历了S-MBP和L-MBP这两种形式的MBP特有的翻译后修饰，并且MBP的胶原样结构域在促进分子组装中起重要作用。
