Characterisation of recombinant glycosylation variants of insulin-like growth factor binding protein-3
Characterisation of recombinant glycosylation variants of insulin-like growth factor binding protein-3
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DOI：
10.1677/joe.0.1600379
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发表时间：
1999-03-01
影响因子：
4
通讯作者：
Baxter, RC
中科院分区：
文献类型：
--
作者：
Firth, SM;Baxter, RC
然后是胰岛素样生长因子结合蛋白-3（IGFBP-3）序列的非保守中心区域中的三个潜在N-糖基化位点（N（89）AS、N（109）AS、（NFS）-F-172）。IGFBP-3以两种糖形式存在，其在酶促去糖基化时还原为单一形式。为了确定碳水化合物链的功能意义，通过用Ala取代Asn残基来单独和组合地突变N-糖基化位点。通过放射免疫测定法检测每个重组糖型，表明糖基化对于中国仓鼠卵巢细胞中的分泌不是必需的。使用[I-125]IGF-I的条件培养基的配体印迹表明所有七种突变体都是活性的。根据检测到的各糖型条带的数量和分子量，Asn（89）、Asn（109）和Asn（172）上分别存在约4、4.5和5 kDa的碳水化合物，Asn（172）占有率可变。在ALS和过量IGF-I存在下，糖变体形成的三元复合物与完全糖基化重组人（rh）IGFBP-3形成的三元复合物无显著差异[K-a（完全糖基化）= 12.5 +/- 4.1 l/nmol;平均K-a（所有突变体）= 22.1 +/- 3.0 l/nmol]。与此相反，天冬氨酸取代天冬氨酸降低酸不稳定亚基（ALS）的结合活性。细胞表面缔合实验表明，糖基化可能会影响IGFBP-3的细胞外环境和细胞表面之间的分区。因此，虽然碳水化合物单元似乎对ALS或IGF结合不是必需的，但它们可能调节IGFBP-3的其他生物活性。
Then are three potential N-glycosylation sites in the non-conserved central region of the insulin-like growth factor binding protein-3 (IGFBP-3) sequence (N(89)AS, N(109)AS, (NFS)-F-172). IGFBP-3 exists as two glyco-forms which reduce to a single form on enzymatic deglycosylation. To determine the functional significance of the carbohydrate chains, the N-glycosylation sites were mutated singly and in combinations by substituting Asn residues with Ala. Each recombinant glycoform was detected by radioimmunoassay, indicating that glycosylation is not essential for secretion in Chinese hamster ovary cells. Ligand blotting of the conditioned media using [I-125]IGF-I indicated that all seven mutants are active. On the basis of the number and molecular masses of the: bands detected for each glycoform, there is approximately 4, 4.5 and 5 kDa of carbohydrate on Asn(89), Asn(109) and Asn(172) respectively, with variable occupancy of Asn(172). Ternary complex formation by the glycovariants in the: presence of ALS and excess IGF-I was not significantly different from that of fully glycosylated recombinant human (rh)IGFBP-3 [K-a (fully glycosylated)= 12.5 +/- 4.1 l/nmol; mean K-a (all mutants) = 22.1 +/- 3.0 l/nmol]. In contrast, Asn to Asp substitutions decreased acid-labile subunit (ALS) binding activity. Cell-surface association experiments indicate that glycosylation may influence the partitioning of IGFBP-3 between the extracellular milieu and the cell surface. Therefore, while the carbohydrate units appear to be non-essential to ALS or IGF binding, they may modulate other biological activities of IGFBP-3.