Elliott S, Bartley T, Delorme E, Derby P, Hunt R, Lorenzini T, Parker V, Rohde M F, Stoney K
Amgen Center, Thousand Oaks, California 91320, USA.
Biochemistry. 1994 Sep 20;33(37):11237-45. doi: 10.1021/bi00203a020.
To define the structural requirements for addition of O-linked glycosylation in vivo, recombinant erythropoietin (rEPO) variants were constructed. Thirty-three independent Ser or Thr substitutions were constructed and examined to see which were subject to O-linked carbohydrate addition. Variants with Thr mutations at positions 123 and 125, but not elsewhere, contained additional carbohydrate, which suggests that several positions around the existing O-linked glycosylation site (Ser126), but not elsewhere, contain the necessary information for O-linked carbohydrate addition. Two forms of the Thr125 variant were identified. One form was glycosylated only at residue 125, and a second form was glycosylated at both Thr125 and Ser126, the normal O-glycosylation site. We have also found that glycosylation is less efficient when rEPO is improperly folded and that prolines at -1 and +1 relative to the O-glycosylation site enhance glycosylation.
为了确定体内O-连接糖基化添加的结构要求，构建了重组促红细胞生成素（rEPO）变体。构建并检测了33个独立的丝氨酸或苏氨酸取代，以确定哪些会发生O-连接碳水化合物添加。在123和125位有苏氨酸突变的变体（其他位置没有）含有额外的碳水化合物，这表明现有O-连接糖基化位点（Ser126）周围的几个位置（而非其他位置）包含O-连接碳水化合物添加所需的信息。鉴定出了两种形式的Thr125变体。一种形式仅在125位残基处糖基化，另一种形式在Thr125和正常O-糖基化位点Ser126处均糖基化。我们还发现，当rEPO折叠不当的时候，糖基化效率较低，并且相对于O-糖基化位点，-1和+1位的脯氨酸会增强糖基化。
