Biosynthesis of surfactant protein D - Contributions of conserved NH2-terminal cysteine residues and collagen helix formation to assembly and secretion
Biosynthesis of surfactant protein D - Contributions of conserved NH2-terminal cysteine residues and collagen helix formation to assembly and secretion
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DOI：
10.1074/jbc.271.31.18912
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发表时间：
1996-08-02
影响因子：
4.8
通讯作者：
Crouch, ED
中科院分区：
文献类型：
--
作者：
BrownAugsburger, P;Chang, D;Crouch, ED
表面活性蛋白 D (SP-D) 优先分泌为十二聚体，由四个通过二硫键交联的胶原三聚体组成。在这些研究中，我们通过稳定转染的 CHO-KI 细胞检查了野生型大鼠 SP-D (RrSP-D) 和选定突变体的生物合成，以确定保守的 N 连接寡糖、胶原螺旋和链间二硫键在 SP-D 组装和分泌中的作用。细胞内形式的 RrSP-D 在 RER 中累积为包含多达四个三聚体亚基的复合物。二硫键交联形成和 RrSP-D 分泌可被 2,2'-联吡啶（脯氨酰和赖氨酰羟化酶抑制剂）和 2 mM 二硫苏糖醇选择性抑制，但不受衣霉素或消除 Asn(70) 糖基化共有序列的影响，尽管用丝氨酸取代 Cys(15) 和 Cys(20) 的突变体(RrSP-Dser15/20) 作为三聚体亚基分泌，具有单个半胱氨酸取代的蛋白质保留在细胞中。令人惊讶的是，RrSP-Dser15/20 的分泌不受2,2'-联吡啶的影响。这些研究强烈表明，SP-D 分泌的最重要和限速步骤涉及交联三聚体亚基的缔合，以形成通过特定亚基间二硫键交联稳定的十二聚体。干扰胶原螺旋形成可通过干扰 NH2 末端结构域的有效二硫键交联来防止分泌。
Surfactant protein D (SP-D) is preferentially secreted as dodecamers consisting of four collagenous trimers cross-linked by disulfide bonds, In these studies, we examined the biosynthesis of wild-type rat SP-D (RrSP-D) and selected mutants by stably transfected CHO-KI cells to determine the roles of a conserved N-linked oligosaccharide, the collagen helix, and interchain disulfide bonds in SP-D assembly and secretion, The major intracellular form of RrSP-D accumulated in the RER as complexes containing up to four trimeric subunits. Disulfide cross-link formation and RrSP-D secretion were selectively inhibited by 2,2'-dipyridyl, an inhibitor of prolyl and lysyl hydroxylase, and by 2 mM dithiothreitol, but unaffected by tunicamycin or elimination of the consensus sequence for glycosylation at Asn(70), Although mutants with serine substituted for Cys(15) and Cys(20) (RrSP-Dser15/20) are secreted as trimeric subunits, proteins with single cysteine substitutions were retained in the cell, Surprisingly, the secretion of RrSP-Dser15/20 was unaffected by 2,2'-dipyridyl. These studies strongly suggest that the most important and rate-limiting step for the secretion of SP-D involves the association of crosslinked trimeric subunits to form dodecamers stabilized by specific inter-subunit disulfide cross-links, Interference with collagen helix formation prevents secretion by interfering with efficient disulfide cross-linking of the NH2-terminal domain.