Meprin A oligomerization contributions of N-linked glycosylation and the MAM domain /

by Ishmael, Susan Senchak.

Abstract (Summary)
iii The oligomerization of proteins can serve to activate or inhibit functional properties of the proteins, create new functions of the oligomeric complex based on subunit composition, concentrate enzymatic activity, and localize the proteins to a particular region of the cell or extracellular space. Meprins are zinc metalloproteases of the astacin family and metzincin superfamily that are obligate oligomers, targeted to the cell surface of brush border epithelial cells in the kidney and intestine, as well as to the surface of certain leukocytes and cancer cell lines. Meprins are composed of two related subunits, ? and ?. Depending on the subunit composition, the meprin oligomers range in size from a membrane-bound, disulfide-bonded dimer (homooligomeric meprin B), to tetramers of heterooligomeric meprin A, to large, secreted ? oligomers of ten or more subunits (homooligomeric meprin A). The work herein investigated the contributions of N-linked glycosylation and the MAM (meprin, A5 protein, protein tyrosine phosphatase ?) domain to the formation and activity of the meprin A homooligomer. Meprins are highly glycosylated, with carbohydrate accounting for approximately 20% of the subunit molecular mass. Using chemical deglycosylation and electrospray mass spectrometry (ESI/MS), it was shown that at least seven of the ten potential N- linked glycosylation sites of mouse meprin A are glycosylated. The glycans are mainly composed of N-acetylglucosamine (GlcNAc), mannose, galactose, and a small amount of fucose. Three glycosylation sites, N234 and N270 in the protease domain and N452 in the TRAF (tumor necrosis factor receptor associated factor) domain, were found to be removed by PNGaseF under native conditions. Removing these glycans did not iv markedly affect the protein conformation or oligomeric state, but decreased the stability of the oligomer as assessed by urea and heat denaturation. A series of single and double glycosylation site mutants were used to determine the contribution of glycosylation to the formation of the meprin oligomer. No one individual glycosylation site affected formation of the oligomer, although one glycosylation site (N41) influenced the amount of secreted protein in the cell culture system. Interestingly, two protease domain glycosylation sites (N152 and N270) were implicated in oligomerization. The N152Q/N270Q and N152Q/N234Q/N270Q mutants were greatly impaired in their ability to form not only the high molecular mass oligomers, but also the disulfide-bonded dimers. These mutations, unlike the other single and double mutants examined, did not retain proteolytic activity, demonstrating that glycosylation of the protease domain is involved in correct tertiary and quaternary structure formation. The noncatalytic domains of meprin, MAM and TRAF, have previously been shown to contribute to oligomerization of meprin A. Here, a series of single point mutants at charged residues in the putative MAM noncovalent interface (K352G, K361Y, R369Q, R376E, D377Y, D378T, R384G, K388L) were created. All mutants were shown to affect formation of the noncovalent interface, with the exception of K361Y. These mutants did not affect disulfide bond formation or proteolytic activity. However, as several variants at position R384 demonstrate, the effect of these residues is not based solely on the presence or absence of charge, but may reflect a strict sequence requirement in the MAM domain of the ? subunit. As these studies demonstrate, charged residues of the MAM domain as well as v posttranslational modifications influence the oligomeric structure of meprin A. However, as these and previous studies have shown, it is unlikely that one particular feature of the ? subunit can be introduced into the ? subunit to promote oligomerization of meprin B to higher order oligomers. This highlights the highly complex folding process that must occur for each meprin subunit.
Bibliographical Information:


School:Pennsylvania State University

School Location:USA - Pennsylvania

Source Type:Master's Thesis



Date of Publication:

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