STUDIES OF THE STRUCTURE OF THE METASTASIS-ASSOCIATED 67- KDA LAMININ-BINDING PROTEIN - FATTY-ACID ACYLATION AND EVIDENCE SUPPORTING DIMERIZATION OF THE 32-KDA GENE-PRODUCT TO FORM THE MATURE PROTEIN
STUDIES OF THE STRUCTURE OF THE METASTASIS-ASSOCIATED 67- KDA LAMININ-BINDING PROTEIN - FATTY-ACID ACYLATION AND EVIDENCE SUPPORTING DIMERIZATION OF THE 32-KDA GENE-PRODUCT TO FORM THE MATURE PROTEIN
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DOI：
10.1021/bi00035a037
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发表时间：
1995-09-05
期刊：
影响因子：
2.9
通讯作者：
STARKEY, JR
中科院分区：
文献类型：
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作者：
LANDOWSKI, TH;DRATZ, EA;STARKEY, JR
67 kDa高亲和力层粘连蛋白结合蛋白（LBP）的表达水平与许多实体瘤的进展相关。67 kDa LBP的cDNA克隆足以编码仅32 kDa的多肽，并且没有容易鉴定的膜结合机制。我们已经过表达转染的67 kDa仓鼠LBP的数量，使我们能够分析膜结合形式的蛋白质。用甲基酯交换试剂处理纯化的LBP，然后用GC-MS鉴定共价结合的脂肪酸棕榈酸酯、硬脂酸酯和油酸酯。脂肪酸修饰可以提供膜缔合的机制。通过MALDI-TOF MS进行的分子量测定表明蛋白质的真实分子量为66.7 kDa，与SDS-PAGE观察到的67 kDa一致。用神经氨酸酶、O-聚糖酶或Endo-F糖苷酶处理LBP对蛋白质的表观分子量没有可检测到的影响，并且MALDI-TOF MS没有显示出通常用糖基化蛋白质观察到的质量异质性的证据。用二硫苏糖醇或β-巯基乙醇还原对SDS-PAGE上的表观分子量或对MALDI-TOF MS上的分子量物质的相对量没有影响。然而，实验测定的氨基酸组成被发现与67 kDa形式是32 kDa前体的同源二聚体一致。初步实验还表明，高亲和力层粘连蛋白结合特性的蛋白质可能是调制的，到目前为止，身份不明的膜辅助分子。
The level of expression of the 67 kDa high-affinity laminin binding protein (LBP) correlates with the progression of many solid tumors. The cDNA clone for the 67 kDa LBP is sufficient to encode a polypeptide of only 32 kDa, and there is no readily identifiable mechanism for membrane association. We have overexpressed the transfected 67 kDa hamster LBP in quantities that have enabled us to analyze the membrane-bound form of the protein. Treatment of the purified LBP with methyl transesterification reagents, follwed by GC-MS, identified the covalently bound fatty acids palmitate, stearate, and oleate. The fatty acid modification may provide a mechanism for membrane association. Molecular mass determination by MALDI-TOF MS demonstrated the true molecular mass of the protein to be 66.7 kDa, compatible with the SDS-PAGE observation of 67 kDa. Treatment of the LBP with neuraminidase, O-glycanase, or Endo-F glycosidase has no detectable effect on the apparent molecular mass of the protein, and the MALDI-TOF MS did not show evidence of mass heterogeneities typically observed with glycosylated proteins. Reduction with dithiothreitol or beta-mercaptoethanol had no effect on the apparent molecular mass on SDS-PAGE or on the relative quantities of molecular mass species on MALDI-TOF MS. The experimentally determined amino acid composition, however, was found to be consistent with the 67 kDa form being a homodimer of the 32 kDa precursor. Preliminary experiments also suggest that the high-affinity laminin binding characteristic of the protein may be modulated by an, as yet, unidentified membrane accessory molecule.