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Functional Roles of N-Linked Glycosylation of Human Matrix Metalloproteinase 9

Tyler Duellman1,2, John Burnett2, Jay Yang1,2

  • 1Molecular and Cellular Pharmacology Graduate Program, University of Wisconsin, School of Medicine and Public Health, Madison, WI, 53705, USA.

Traffic (Copenhagen, Denmark)
|July 25, 2015
PubMed

Insights

N-glycosylation regulates human matrix metalloproteinase-9 (hMMP-9) secretion by modulating its interaction with calreticulin (CALR). This study reveals a novel mechanism where N-glycosylation at residue 38 influences CALR binding, impacting hMMP-9 secretion.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Matrix metalloproteinase-9 (MMP-9) is crucial for extracellular matrix regulation and signaling.
  • Human MMP-9 (hMMP-9) possesses N-glycosylation sites at N38 and N120, but their functional roles are largely unknown.
  • Expression of N-glycosylation-deficient hMMP-9 variants has been hindered by miRNA-mediated inhibition.

Purpose of the Study:

  • To investigate the role of N-glycosylation at residues N38 and N120 in hMMP-9 secretion.
  • To elucidate the mechanism by which N-glycosylation impacts hMMP-9 secretion.
  • To overcome challenges in expressing N-glycosylation-deficient hMMP-9 variants.

Main Methods:

  • Engineered hMMP-9 cDNA with amino acid substitutions (mS38, N120S) in a miRNA-binding site disrupted template.
  • Expressed modified hMMP-9 variants via transient transfection.
  • Analyzed protein secretion, endoplasmic reticulum (ER) retention using imaging, and protein interactions via co-immunoprecipitation with calreticulin (CALR).

Main Results:

  • A single mS38 substitution supported good secretion, while N120S or double mS38/N120S mutations significantly reduced secretion.
  • Non-secreted variants exhibited ER retention and enhanced interaction with the ER-resident protein CALR.
  • Removal of N-glycosylation at residue 38 exposed an N-glycosylation-independent interaction with CALR, likely causing ER retention.

Conclusions:

  • N-glycosylation critically regulates hMMP-9 secretion.
  • This regulation involves a novel mechanism where N-glycosylation influences an N-glycosylation-independent interaction with CALR, affecting protein trafficking.
  • The findings provide new insights into glycoprotein secretion and ER quality control pathways.
Keywords:
ER retentionN-glycosylationcalreticulinco-IP assaycomplementation assaymatrix metalloproteinase-9molecular volumemutagenesissecretion

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