Identification of Domains and Amino Acids Essential to the Collagen Galactosyltransferase Activity of GLT25D1

Collagen is modified by hydroxylation and glycosylation of hydroxylysine residues. This glycosylation is initiated by the β1,O galactosyltransferases GLT25D1 and GLT25D2. The structurally similar protein cerebral endothelial cell adhesion molecule CEECAM1 was previously reported to be inactive when assayed for collagen glycosyltransferase activity. To address the cause of the absent galactosyltransferase activity, we have generated several chimeric constructs between the active human GLT25D1 and inactive human CEECAM1 proteins. The assay of these chimeric constructs pointed to a short central region and a large C-terminal region of CEECAM1 leading to the loss of collagen galactosyltransferase activity. Examination of the three DXD motifs of the active GLT25D1 by site-directed mutagenesis confirmed the importance of the first (amino acids 166–168) and second motif (amino acids 461–463) for enzymatic activity, whereas the third one was dispensable. Since the second DXD motif is incomplete in CEECAM1, we have restored the motif by introducing the substitution S461D. This change did not restore the activity of the C-terminal region, thereby showing that additional amino acids were required in this C-terminal region to confer enzymatic activity. Finally, we have introduced the substitution Q471R-V472M-N473Q-P474V in the CEECAM1-C-terminal construct, which is found in most animal GLT25D1 and GLT25D2 isoforms but not in CEECAM1. This substitution was shown to partially restore collagen galactosyltransferase activity, underlining its importance for catalytic activity in the C-terminal domain. Because multiple mutations in different regions of CEECAM1 contribute to the lack of galactosyltransferase activity, we deduced that CEECAM1 is functionally different from the related GLT25D1 protein.胶原蛋白（Collagen）可通过羟赖氨酸残基的羟化作用与糖基化作用完成修饰。该糖基化过程由β1,O-半乳糖基转移酶（β1,O galactosyltransferases）GLT25D1与GLT25D2启动。结构相似的脑内皮细胞黏附分子（cerebral endothelial cell adhesion molecule, CEECAM1）此前被报道在胶原蛋白糖基转移酶活性检测中呈现无活性状态。 为明确其半乳糖基转移酶活性缺失的原因，我们构建了活性人源GLT25D1与无活性人源CEECAM1之间的多组嵌合蛋白构建体。对这些嵌合构建体的活性分析表明，CEECAM1的一段短小中心区域与大型C端区域是导致其丧失胶原蛋白半乳糖基转移酶活性的关键区段。 通过定点诱变（site-directed mutagenesis）技术对活性GLT25D1的三个DXD基序（DXD motif）进行功能验证，结果证实第一个基序（氨基酸残基166–168位）与第二个基序（氨基酸残基461–463位）对酶促活性至关重要，而第三个DXD基序则非必需。由于CEECAM1中的第二个DXD基序并不完整，我们通过引入S461D氨基酸替换修复了该基序，但这一改动并未恢复其C端区域的酶活性，这说明该C端区域还需要额外的氨基酸残基才能赋予其催化活性。 最后，我们在CEECAM1的C端构建体中引入了Q471R-V472M-N473Q-P474V氨基酸替换组合，该序列组合存在于绝大多数动物源GLT25D1与GLT25D2同工型（isoforms）中，但未在CEECAM1中发现。实验结果显示，该替换组合可部分恢复胶原蛋白半乳糖基转移酶活性，凸显了其在C端结构域催化活性中的重要性。 鉴于CEECAM1不同区域的多处突变均会导致其半乳糖基转移酶活性缺失，我们推断CEECAM1在功能上与同源的GLT25D1蛋白存在显著差异。