PetersPlus综合征(PTRPLSOMIM#261540)是一种严重的先天性糖基化疾病,患者有多个结构异常,包括眼睛的Peters异常(眼前节发育不全),不成比例的身材矮小,Brachydactyly,畸形面部特征,发育迟缓,和可变的额外异常。PTRPLS患者和一些PetersPlus样(PTRPLS样)患者(仅具有PTRPLS表型的子集)在编码β1,3-葡萄糖基转移酶(B3GLCT)的基因中具有突变。B3GLCT在血小板反应蛋白1型重复序列上催化葡萄糖向O-连接的岩藻糖的转移。大多数B3GLCT底物蛋白属于ADAMTS超家族,在细胞外基质中起关键作用。我们试图确定PTRPLS或PTRPLS样突变是否消除B3GLCT活性。B3GLCT有两个假定的活性位点,一个在N末端区域,另一个在C末端糖基转移酶结构域。使用序列分析和体外活性测定,我们证明了C-末端结构域催化葡萄糖向O-连接的岩藻糖的转移。我们还建立了B3GLCT的同源模型,并将D421鉴定为催化碱基。PTRPLS和PTRPLS样突变分别导入B3GLCT,并且使用体外酶测定和基于细胞的功能测定来评估突变的酶。我们的结果表明PTRPLS突变导致B3GLCT酶活性丧失和/或显著降低蛋白质稳定性。相比之下,具有PTRPLS样突变的B3GLCT保留了酶活性,尽管有些显示出轻微的不稳定作用。总的来说,我们的数据支持以下假设:B3GLCT底物蛋白的葡萄糖丢失是PTRPLS患者中观察到的缺陷的原因,但不适用于PTRPLS样患者。
Peters Plus Syndrome (PTRPLS OMIM #261540) is a severe congenital disorder of glycosylation where patients have multiple structural anomalies, including Peters anomaly of the eye (anterior segment dysgenesis), disproportionate short stature, brachydactyly, dysmorphic facial features, developmental delay, and variable additional abnormalities. PTRPLS patients and some Peters Plus-like (PTRPLS-like) patients (who only have a subset of PTRPLS phenotypes) have mutations in the gene encoding β1,3-glucosyltransferase (B3GLCT). B3GLCT catalyzes the transfer of glucose to O-linked fucose on thrombospondin type-1 repeats. Most B3GLCT substrate proteins belong to the ADAMTS superfamily and play critical roles in extracellular matrix. We sought to determine whether the PTRPLS or PTRPLS-like mutations abrogated B3GLCT activity. B3GLCT has two putative active sites, one in the N-terminal region and the other in the C-terminal glycosyltransferase domain. Using sequence analysis and in vitro activity assays, we demonstrated that the C-terminal domain catalyzes transfer of glucose to O-linked fucose. We also generated a homology model of B3GLCT and identified D421 as the catalytic base. PTRPLS and PTRPLS-like mutations were individually introduced into B3GLCT, and the mutated enzymes were evaluated using in vitro enzyme assays and cell-based functional assays. Our results demonstrated that PTRPLS mutations caused loss of B3GLCT enzymatic activity and/or significantly reduced protein stability. In contrast, B3GLCT with PTRPLS-like mutations retained enzymatic activity, although some showed a minor destabilizing effect. Overall, our data supports the hypothesis that loss of glucose from B3GLCT substrate proteins is responsible for the defects observed in PTRPLS patients, but not for those observed in PTRPLS-like patients.