The Cytoplasmic Tail Dileucine Motif LL572 Determines the Glycosylation Pattern of Membrane-type 1 Matrix Metalloproteinase

Thomas Ludwig,Sarah M Theissen,Michael J Morton,Michael J Caplan

doi:10.1074/jbc.m801816200

Abstract

Membrane-type 1 matrix metalloproteinase (MT1-MMP; MMP-14) drives fundamental physiological and pathological processes, due to its ability to process a broad spectrum of substrates. Because subtle changes in its activity can produce profound physiological effects, MT1-MMP is tightly regulated. Currently, many aspects of this regulation remain to be elucidated. It has recently been discovered that O-linked glycosylation defines the substrate spectrum of MT1-MMP. We hypothesized that a mutual interdependency exists between MT1-MMP trafficking and glycosylation. Lectin precipitation, metabolic labeling, enzymatic deglycosylation, and site-directed mutagenesis studies demonstrate that the LL(572) motif in the cytoplasmic tail of MT1-MMP influences the composition of the complex O-linked carbohydrates attached to the hinge region of the protein. This influence appears to be independent from major effects on cell surface trafficking. MT1-MMP undergoes extensive processing after its synthesis. The origins and the molecular characters of its multiple forms are incompletely understood. Here, we develop and present a model for the sequential, post-translational processing of MT1-MMP that defines stages in the post-synthetic pathway pursued by the protein.

Highlights

Analysis of potential N-glycosylation sites in Membrane-type 1 matrix metalloproteinase (MT1-Matrix metalloproteinases (MMPs)) with the NetNGlyc server software revealed the absence of the canonical Asn-Xaa-Ser/Thr N-glycosylation sequence, indicating that this protein cannot be subject to this post-translational modification
Because the electrophoretic mobilities of the MT1-MMP putative glycosylation mutants CHO-4 and CHO-5 did not differ from one another (Fig. 2a) we conclude that Ser304 is not glycosylated
Insight into their function arrow 3) that corresponds to the calculated molecular mass of in diverse biological processes will require the elucidation of active MT1-MMP without the pro-domain

Summary

EXPERIMENTAL PROCEDURES

General Reagents—Unless otherwise noted, all reagents were purchased from Sigma-Aldrich. Lectin Precipitation Assay—Cells were transfected with double tagged MT1-MMP constructs and lysed according to the protocol described above. COS cells were transfected with double tagged MT1MMP constructs and incubated for 48 h under tissue culture conditions at a 40 ␮M final concentration of Ac4-GalNAz (C33365, Invitrogen). Transfected cells were lysed, and 10 ␮l of lysate was mixed and incubated with 3 ␮l of 5ϫ reaction buffer and 2 ␮l of sialidase A (activity Ͼ 5 units/ml) or solvent control for 12 h at 37 °C. Cells were placed on ice after 30 min and washed twice with ice-cold chase media (minimal essential medium supplemented with 10% fetal bovine serum and 10 mM L-methionine and 10 mM L-cysteine, pH 7.4, at 5% CO2). Lysates of untransfected cells and lysates of transfected cells without addition of precipitating Ab were used as controls (Fig. 5)

RESULTS

It corresponds essentially to the

DISCUSSION