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

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 ϳ64 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.

The Collagen Binding Domain of Gelatinase A Modulates Degradation of Collagen IV by Gelatinase B

“…4 The secreted pro-MMP9 was purified to homogeneity by gelatin-Sepharose chromatography 56 and dialyzed into 100 mM Tris-HCl, pH 7.4, 100 mM NaCl and 10 mM CaCl 2 . The proenzyme was activated by incubating 10 μM progelatinase solution with a solution of 0.1 μM cdMMP3 (Biomol International, USA) at 37°C for 90 min.…”

“…Furthermore, only in the case of MMP9 is the hinge region a long O-glycosylated domain. 4 Although most MMPs have similar substrate specificities, their redundant activities are kept separated by tissue distribution and regulation. 5 In particular, gelatinases (i.e., MMP2 and MMP9) are normally compartmentalized, with MMP2 being present in blood serum at high concentrations (up to 160 ng/ml) 6 and MMP9 being mostly stored in neutrophil granules.…”

Enzymatic processing of β‐dystroglycan recombinant ectodomain by MMP‐9: Identification of the main cleavage site

“…Recombinant human full length MMP-9 was produced in Sf9 insect cells after transfection with a baculovirus carrying the MMP-9 cDNA (27). The secreted proMMP-9 was purified to homogeneity by gelatin-Sepharose chromatography (28) and dialyzed against 100 mM Tris/HCl pH 7.4, 100 mM NaCl, 10 mM CaCl 2 .…”

Enzymatic processing by MMP‐2 and MMP‐9 of wild‐type and mutated mouse β‐dystroglycan

“…The applied gradient was 0-4 min 5% B, 4-34 min from 5 to 50% B (linear), and 34-64 min from 50 to 90% B (linear) at a flow rate of 80 lL min 21 . In data-dependent acquisition mode, the three most intense multicharged ions were selected and fragmented by using collision-induced dissociation (35% normalized collision energy) and then the spectra were recorded.…”

“…The intrinsic structural features of the two gelatinases could furnish an explanation for such a divergent interaction with the same substrate. The highly glycosylated OG domain of MMP-9 confers flexibility to the enzyme and makes it suitable to interact with large macromolecular substrates such as collagen (21,27,28), although the OG domain presumably limits the accessibility to small substrates such as the b-DG ectodomain. Therefore, in a pathological context, as in the case of a tumoral microenvironment, the smaller structural arrangement of MMP-2 seems to be more suitable for the b-DG ectodomain breakdown.…”