HOMOLOGOUS MEMBRANE FOLATE BINDING-PROTEINS IN HUMAN-PLACENTA - CLONING AND SEQUENCE OF A CDNA
HOMOLOGOUS MEMBRANE FOLATE BINDING-PROTEINS IN HUMAN-PLACENTA - CLONING AND SEQUENCE OF A CDNA
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DOI：
10.1021/bi00446a042
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发表时间：
1989-10-03
期刊：
影响因子：
2.9
通讯作者：
FREISHEIM, JH
中科院分区：
文献类型：
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作者：
RATNAM, M;MARQUARDT, H;FREISHEIM, JH
从人胎盘膜中纯化的叶酸结合蛋白，在多种蛋白酶抑制剂的存在下，用n -聚糖酶去糖基化，在Mr .apprx上有一个尖锐的条带。28000个sds -聚丙烯酰胺凝胶电泳。去糖基化蛋白与[3H]叶酸的结合与天然蛋白一样紧密。用葡萄球菌V8蛋白酶对纯化蛋白进行酶切和HPLC纯化后获得的肽段进行测序。对该蛋白制备的多克隆抗体进行亲和纯化，用于筛选胎盘cDNA表达文库。由此获得了胎盘叶酸结合蛋白的全长cDNA，并推导出相应的蛋白序列。这一结果，结合肽序列数据，表明至少有两个同源叶酸结合蛋白在胎盘中表达，其中一个似乎与人乳和鼻咽部表皮样癌（KB）细胞中的叶酸结合蛋白相同；得到的cDNA序列与其他蛋白相对应。推导出的蛋白质序列的特征是假定的16个残基的氨基末端信号肽被切割，从而产生239个残基的多肽。成熟蛋白在Asn-99和Asn-179上有两个潜在的n链糖基化位点，8个潜在的分子内二硫键，在羧基端有一段可以形成跨膜结构域的疏水残基。该蛋白与KB细胞叶酸结合蛋白有68%的序列同源性，可能代表胎儿叶酸转运蛋白。本文讨论了支持以下论点的证据：各种组织中的可溶性叶酸结合蛋白不仅仅是膜蛋白蛋白水解加工的产物，而且这两种蛋白必须通过某种类型的共价修饰而有所不同。
A preparation of folate binding protein purified from human placental membranes in the presence of a variety of protease inhibitors followed by deglycosylation with N-glycanase gave a sharp band at Mr .apprx. 28,000 following SDS-polyacrylamide gel electrophoresis. The deglycosylated protein bound [3H]folic acid as tightly as the native protein. Peptides obtained following digestion of the purified protein with staphylococcal V8 protease and HPLC purification were sequenced. Polyclonal antibodies against the protein preparation were affinity purified and used to screen a placental cDNA expression library. A full-length cDNA for a placental folate binding protein was thus obtained and the corresponding protein sequence deduced. This result, taken together with the peptide sequence data, indicates the expression of at least two homologous folate binding proteins in placenta, one of which appears to be identical with the folate binding protein from human milk and nasopharyngeal epidermoid carcinoma (KB) cells; the cDNA sequence obtained corresponds to the other proteins. The deduced protein sequence is characterized by a putative 16-residue amino-terminal signal peptide that is cleaved, resulting in a 239-residue polypeptide. The mature protein exhibits two potential sites of N-linked glycosylation at Asn-99 and Asn-179, eight potential intramolecular disulfide bonds, and a stretch of hydrophobic residues at the carboxyl terminus that could form a transmembrane domain. The protein bears a 68% sequence homology with the KB cell folate binding protein and may represent a fetal folate transport protein. Evidence is discussed in support of the contention that the soluble folate binding protein in various tissues is not simply a product of proteolytic processing of the membrane protein and that the two proteins must differ by some type(s) of covalent modification.