New insights into the catalytic inactivity of mammary gland protein-40, a chitinase-like protein expressed during mammary gland involution.

MGP-40 is a mammary gland-specific glycoprotein which is expressed during involution and is an important marker for mammary gland apoptosis. It is an inactive chitinase-like protein belonging to Glycosyl Hydrolase family 18. The present study reports sequence characterization, tissue-specific expression analysis, production of recombinant MGP-40 and its mutant (A117D and L119E) in both E. coli and COS1 cells for their chitin-binding and chitinase activity analysis. The cDNA of buffalo MGP-40 was cloned and sequenced which corresponded to 1803 bp with an open reading frame of 1152 bp (361 aa), signal sequence of 63 bp (21 aa), 5' and 3' UTR of 144 bp and 507 bp, respectively. The 3' UTR analysis revealed potential sites for high level expression and stability during involution. The half-life of buffalo MGP-40 was found to be 11.7 h. MGP-40 was highly expressed in mammary gland followed by small intestine, spleen and mammary epithelial cells. The purified recombinant MGP-40 and its mutant expressed in E.coli were observed to bind chitin efficiently, however, no chitinase activity was observed. Further, chitinase activity was also not observed by expressing mutant recombinant MGP-40 in COS1 cells ruling out the possible role of post-translational modifications. Structure-based in-silico mutagenesis by FoldX algorithm showed a drastic decrease in overall fold stability which might be a possible reason for inability to recover its activity. Therefore, chitinase activity could not be restored in MGP-40 even after reverting back two critical residues in active site which may be due to detrimental effect of mutations on structural stability.