The Hemopexin and O-Glycosylated Domains Tune Gelatinase B/MMP-9 Bioavailability via Inhibition and Binding to Cargo Receptors

Philippe E Van Den Steen,Ilse Van Aelst,Vibeke Hvidberg,Helene Piccard,Pierre Fiten,Christian Jacobsen,Soren K Moestrup,Simon Fry,Louise Royle,Mark R Wormald,Russell Wallis,Pauline M Rudd,Raymond A Dwek,Ghislain Opdenakker

doi:10.1074/jbc.m512308200

Abstract

Gelatinase B/matrix metalloproteinase-9 (MMP-9), a key regulator and effector of immunity, contains a C-terminal hemopexin domain preceded by a unique linker sequence of approximately 64 amino acid residues. This linker sequence is demonstrated to be an extensively O-glycosylated (OG) domain with a compact three-dimensional structure. The OG and hemopexin domains have no influence on the cleavage efficiency of MMP-9 substrates. In contrast, the hemopexin domain contains a binding site for the cargo receptor low density lipoprotein receptor-related protein-1 (LRP-1). Furthermore, megalin/LRP-2 is identified as a new functional receptor for the hemopexin domain of MMP-9, able to mediate the endocytosis and catabolism of the enzyme. The OG domain is required to correctly orient the hemopexin domain for inhibition by TIMP-1 and internalization by LRP-1 and megalin. Therefore, the OG and hemopexin domains down-regulate the bioavailability of active MMP-9 and the interactions with the cargo receptors are proposed to be the original function of hemopexin domains in MMPs.

Highlights

Natural MMP-9 produced by neutrophils is a heavily glycosylated protein
This corresponds with the known primary sequence preferences of O-linked glycosylation, for which a Pro is preferred one position in front of or 3 positions behind Ser or Thr [45], as the OG domain is composed of 12 repeats of the sequence (Thr/Ser)-Xxx-Xxx-Pro
In natural MMP-9 isolated from human neutrophils, the O-linked sugars consist of the classical core 1 (Gal-GalNAc-O-Ser) or core 2 (Gal-(GlcNAc-)GalNAc-O-Ser) structures, which are further elongated to relatively large glycans, consisting of up to 10 monosaccharide residues [10]

Summary

EXPERIMENTAL PROCEDURES

The cDNA of human MMP-9, a kind gift from Dr G. MMP-9 variants (10 ␮M) were activated with 0.1 ␮M recombinant catalytic domain of human stromelysin-1 (Calbiochem), without the presence of APMA, during 90 min at 37 °C in assay buffer (100 mM Tris/HCl, pH 7.4, 100 mM NaCl, 10 mM CaCl2). To estimate the cleavage efficiency of various substrates by the different MMP-9 variants, denatured bovine collagen II [24], MMP-8cleaved native collagen II [25], ␣1-antitrypsin inhibitor (R&D Systems, Abingdon, UK; 0.5 ␮M) [26], mouse eye extract containing ␤B1-crystallin [27], recombinant intact mouse granulocyte chemotactic protein-2 (mGCP-2/LIX, Peprotech Inc, Rocky Hill, NJ; 2 ␮M) [28], recombinant interleukin-8 (IL-8, Peprotech, 2 ␮M) [29], galectin-3 (R&D Systems, 30 ␮g/ml) [30], soluble recombinant ICAM-1/Fc chimera (R&D Systems, 30 ␮g/ml) [31], soluble recombinant CD25/Fc chimera (R&D Systems, 30 ␮g/ml) [32], human plasminogen (R&D Systems, 30 ␮g/ml) [33], human platelet SPARC (Calbiochem, 1 ␮M) [34] or recombinant MCP-3 (Peprotech, 0.5 ␮M) was incubated in 100 mM Tris/HCl pH 7.4, 100 mM NaCl, 10 mM CaCl2 with different concentrations of activated MMP-9 variants at 37 °C. Kinetic parameters were determined by BIAevaluation 4.1 software using a Langmuir 1:1 binding model and simultaneous fitting of all curves in a concentration range from 5–500 nM (global fitting)

Endocytosis Analysis

Analytical Ultracentrifugation

Molecular Modeling

RESULTS

Gelatin II

DISCUSSION