PURIFICATION, CHARACTERIZATION AND CDNA CLONING OF HUMAN LUNG SURFACTANT PROTEIN-D
PURIFICATION, CHARACTERIZATION AND CDNA CLONING OF HUMAN LUNG SURFACTANT PROTEIN-D
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DOI：
10.1042/bj2840795
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发表时间：
1992-06-15
影响因子：
4.1
通讯作者：
REID, KBM
中科院分区：
文献类型：
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作者：
LU, JH;WILLIS, AC;REID, KBM
人肺表面活性蛋白 D (SP-D) 在肺灌洗中通过其分子量和与麦芽糖的 Ca2+ 依赖性结合亲和力与大鼠 SP-D 的相似性而被鉴定 [Persson, Chang & Crouch (1990) J. Biol.化学。 265、5755-5760]。为了进行结构研究，通过麦芽糖-琼脂糖凝胶上的亲和层析和 f.p.l.c. 从羊水中分离出人 SP-D。 Superose 6 上显示它的分子量约为。非解离条件下为 620 kDa。在 SDS/PAGE 上，人 SP-D 在非还原或还原条件下分别表现为 150 kDa 或 43 kDa 的单条带。人SP-D的氨基酸组成中存在高浓度的甘氨酸(22%)、羟脯氨酸和羟赖氨酸，表明它含有类胶原蛋白结构。胶原酶消化产生 20 kDa 的抗胶原酶球状片段，该片段保留了对麦芽糖的亲和力。在固相结合测定中使用麦芽糖基-BSA作为新糖蛋白配体表明，人 SP-D 与大鼠 SP-D 具有相似的碳水化合物结合特异性，但与其他凝集素（例如羽扇豆凝集素）对一系列简单糖的特异性明显不同。氨基酸序列分析确定了人 SP-D 中存在胶原蛋白样 Gly-Xaa-Yaa 三联体，并且还提供了用于合成寡核苷酸探针的分子球状区域的序列数据。使用寡核苷酸探针以及兔抗（人SP-D）筛选人肺cDNA文库，允许分离两个重叠的cDNA克隆，从而给出人SP-D的完整编码序列。推导的氨基酸序列表明，成熟的人SP-D多肽链长355个残基，具有25个残基的短非胶原样N末端部分，随后是177个残基的胶原样区域和153个残基的C末端C型凝集素结构域。人 SP-D 和牛血清球凝素氨基酸序列的比较表明，尽管它们在碳水化合物特异性方面存在显着差异，但它们显示出 66% 的同一性。
Human pulmonary surfactant protein D (SP-D) was identified in lung lavage by its similarity to rat SP-D in both its molecular mass and its Ca2+-dependent-binding affinity for maltose [Persson, Chang & Crouch (1990) J. Biol. Chem. 265, 5755-5760]. For structural studies, human SP-D was isolated from amniotic fluid by affinity chromatography on maltose-Sepharose followed by f.p.l.c. on Superose 6, which showed it to have a molecular mass of approx. 620 kDa in non-dissociating conditions. On SDS/PAGE the human SP-D behaved as a single band of 150 kDa or 43 kDa in non-reducing or reducing conditions respectively. The presence of a high concentration of glycine (22 %), hydroxyproline and hydroxylysine in the amino acid composition of human SP-D indicated that it contained collagen-like structure. Collagenase digestion yielded a 20 kDa collagenase-resistant globular fragment which retained affinity for maltose. Use of maltosyl-BSA as a neoglycoprotein ligand in a solid-phase binding assay showed that human SP-D has a similar carbohydrate-binding specificity to rat SP-D, but a clearly distinct specificity from that of other lectins, such as conglutinin, for a range of simple saccharides. Amino acid sequence analysis established the presence of collagen-like Gly-Xaa-Yaa triplets in human SP-D and also provided sequence data from the globular region of the molecule which was used in the synthesis of oligonucleotide probes. Screening of a human lung cDNA library with the oligonucleotide probes, and also with rabbit anti-(human SP-D), allowed the isolation of two cDNA clones which overlap to give the full coding sequence of human SP-D. The derived amino acid sequence indicates that the mature human SP-D polypeptide chain is 355 residues long, having a short non-collagen-like N-terminal section of 25 residues, followed by a collagen-like region of 177 residues and a C-terminal C-type lectin domain of 153 residues. Comparison of the human SP-D and bovine serum conglutinin amino acid sequences indicated that they showed 66 % identity despite their marked differences in carbohydrate specificity.