cDNA cloning of milk fat globule membrane-glycoprotein antigens recognized by the monoclonal antibodies

乳脂肪球膜（MFGM）起源于哺乳期乳腺上皮细胞的质膜，含有多种膜糖蛋白。这些MFGM糖蛋白的结构和生物学功能尚未得到很好的表征。本研究以泌乳奶牛乳腺组织mRNA为模板构建了MFGM糖蛋白cDNA文库，并以MFGM单克隆抗体为探针进行了筛选。选择几个阳性克隆并用作PCR扩增的模板。对部分克隆进行了序列测定，推导的氨基酸序列表明，其中一个克隆编码的cDNA与从小鼠乳腺中克隆的小鼠cDNA MFG-E8相似。利用该cDNA片段通过RT-PCR克隆cDNA的5'和3'末端，并测定全长cDNA的核苷酸序列。推导的氨基酸序列表明，被MFGM糖蛋白抗原MGP 53/57特异性的单克隆抗体3F 12识别的一个克隆与小鼠MFG-E8相似，并含有MGP 53/57的一些氨基酸序列。因此，发现该cDNA编码主要MFGM糖蛋白MGP 53/57。MGP 53/57也具有EGF样结构域和与因子VIII C区同源的重复结构，但缺少MFG-E8序列中的富含脯氨酸区，推测的氨基酸序列中存在3个潜在的N-糖基化位点。MGP 53/57的53 kDa和57 kDa的两种异构体被认为是由于单个多肽的每个糖基化位点的差异糖基化。

Milk fat globule membrane (MFGM) is originated from plasma membrane of lactating mammary epithelial cells, and contains a variety of membrane glycoproteins. Structure and biological functions of these MFGM glycoproteins have not yet been well characterized. The objective of this research was to isolate cDNA clones and determine their nucleotide sequences to characterize protein structure and presume unknown functions of the MFGM glycoprotein antigens recognized by the monoclonal antibodies raised against MFGM.A cDNA library was constructed from mRNA of mammary gland of a lactating cow, and screened by using monoclonal antibodies as probes. Several positive clones were selected and used as template for PCR amplification. Some clones were sequenced and the deduced amino acid sequences indicated that one of these clones encoded cDNA similar to the mouse cDNA MFG-E8 cloned from mouse mammary gland. The 5' and 3' ends of the cDNA were cloned by RT-PCR using the cDNA fragment, and the nucleotide sequence for a full lenght cDNA was determined. The deduced amino acid sequence showed that one clone recognized by the monoclonal antibody 3F12, which is specific to MFGM glycoprotein antigen MGP53/57, was similar to mouse MFG-E8, and contained some amino acid sequences determined for MGP53/57. Thus, this cDNA was found to encode the major MFGM glycoprotein MGP53/57. MGP53/57 also has a EGF-like domain and a repeat-sturucture homologous to the C region of Factor VIII,but lacks the proline-rich region which was found in the MFG-E8 sequence.There were three potential N-glycosylation sites in the deduced amino acid sequence of MGP53/57. Two isoforms with 53kDa and 57kDa of MGP53/57 was suggested to be due to differential glycosylation to each glycosylation site fo the single polypeptide.