Alternative splicing of MAGP2 generates a non‐glycosylated variant still capable of localizing to extracellular microfibrils
Authors: Miyamoto Alison R. 
Published: June 2021

Alterations in microfibril‐associated glycoprotein 2 (MAGP2) have been linked to familial thoracic aortic aneurysm and dissection (FTAAD) and ovarian cancer, suggesting that this protein has an interesting role in normal adult homeostasis. Here, a previously unstudied splice variant of MAGP2 noted in a public database search was characterized. Two mRNA isoforms of MAGP2 were expressed in a human ovarian cancer cell line (CAOV3) by RT‐PCR; sequencing revealed that the longer mRNA was the canonical MAGP2 mRNA that encodes a 173‐amino acid protein (MAGP2‐a), and the shorter variant encoded a 163‐amino acid protein (MAGP2‐b), due to the removal of the seventh exon. Interestingly, the seventh exon contains the conserved asparagine residue (Asp79) thought to be the major site of N‐glycosylation on MAGP2. As expected, MAGP2‐b was both poorly glycosylated and poorly secreted when overexpressed in either 293T cells or a mouse ovarian epithelial cell line, T3, as measured by immunoblotting analysis; this result is consistent with the canonical role of glycosylation modifications of secreted proteins. Since a small fraction of MAGP2‐b protein was secreted, studies were performed to understand what functions MAGP2‐b was capable of, if secreted. To determine if MAGP2‐b could bind and localize to extracellular microfibrils as previously reported for MAGP2‐a, immunofluorescence studies were conducted in T3 cells that generate a striking fibrillin‐2‐rich microfibril matrix in cell culture. Surprisingly, MAGP2‐b could easily be found localized to microfibrils, at levels similar to the MAGP2‐a isoform that is glycosylated and more effectively secreted than MAGP2‐b. To more clearly delineate the functional consequences of glycosylation versus the loss of ten amino acids via alternative splicing, an Asp79Gln (N79Q) mutation was generated in MAGP2‐a to block N‐glycosylation. The N79Q MAGP2‐a protein does not appear to be glycosylated, since the molecular weight of the protein on immunoblots is unchanged by PNGase F treatment. The secretion and microfibril binding properties of the N79Q mutant are very similar to MAGP2‐b; they are both poorly secreted but still able to bind to microfibrils at levels equal to or greater than the fully glycosylated MAGP2‐a variant. Together, these data indicate that glycosylation of MAGP2 is not required for microfibril binding; it also suggests that the non‐glycosylated MAGP2‐b may preferentially bind to microfibrils after cellular export. Why might it be useful for such a MAGP2 variant to exist? In this regard, there is some correlative evidence to suggest that MAGP2 binding of active TGF‐β may negatively regulate TGF‐β activity, based on studies on MAGP2 biochemistry and FTAAD‐associated mutations of MAGP2. Although speculative, the existence of a mechanism that tethers MAGP2 to microfibrils at the expense of free, soluble MAGP2 may make MAGP2 a more effective microfibril tether for active TGF‐β.Support or Funding InformationCSUF Department of Biological Science. Student research assistants funded by CIRM Bridges to Stem Cell Research (Andrew Yale, Cherie Petersen) and HHMI Undergraduate Research Program (Ryan Daniels).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.