|Budget Amount *help
¥2,600,000 (Direct Cost : ¥2,600,000)
Fiscal Year 1996 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1995 : ¥1,800,000 (Direct Cost : ¥1,800,000)
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.