Title

Heparin/heparan sulfate proteoglycans are potential extracellular docking molecules for matrix metalloproteases and tissue inhibitors of metalloprotease-3

Date of Award

1999

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

J. Frederick Jr. Woessner, Committee Chair

Abstract

Transient upregulation of heparan/chondroitin sulfate proteoglycan has been reported in the edge of the growth cone of the out-growth neurite, epidermal basement membrane of the wound area, and pseudopods of the invading cells, such as metastatic cancer cells and extravasated monocytes/macrophages. In this report, we present a novel significant role for these highly negative sulfated sugar polymers: they function as extracellular docking molecules for matrix metalloproteases, (MMPs) and TIMP-3. The smallest MMP, matrilysin (MMP-7), has the minimum structure, but anchors firmly on the cell surface or in the extracellular matrix. Enzyme can be extracted from times with heparin, sulfated glycosaminoglycans and related compounds and by heparinase treatment. Heparan sulfate chains on the cell surface and in the basement membrane are suggested to be the most likely docking molecules. Collagenase 1 and 3 and gelatinase A and B are similarly, but more weakly, bound. Heparin binding of recombinant rat proMMP-7 was examined by affinity chromatography, affinity coelectrophoresis, and homogeneous enzyme-based binding assay, the KD is 5--10 nM. Zymographic measurement of MMP-1, -7 and -13 is greatly enhanced by heparin which suggests that this docking molecule could also function as an MMPs enhancer in vivo. Recombinant proMMP-7 is able to bind to the GAG modified form of CD44 (Epican/CD44v3-10) transfected into a mammalian cell line. Two heparin-binding peptides have been identified, matching C- and N-terminal regions of proMMP-7, but molecular modeling suggests a more extensive binding track or cradle extending across many strands on the face opposite the active center. Such binding presents a novel method for regulating enzyme amount, activation and orientation with respect to the cell surface and my be of importance in cancer progression.The in vivo extraction of TIMP-3 and the in vitro heparin-binding assays for recombinant TIMP-3 reveals that TIMP-3 binds to many sulfated glycosaminoglycans but this binding is of lower affinity compared to matrilysin. N-TIMP-3 binds to heparin affinity chromatography columns with the same affinity as full length TIMP-3. A potential heparin-binding AB loop was identified in TIMP-3. A novel MMP inhibitory protein with molecular weight 16 kDa was found in postpartum uterus, spleen, kidney, king and intestine by using heparin, heparan sulfate, pentosan sulfate and suramin sulfate as extractants. This protein is not in the TIMP nor PCP enhancer family.The recombinant catalytic zinc binding domain (ZBD), residues 188--247 of MMP-7, catalyzed the hydrolysis of a peptide substrate with 400 ∼ 500 fold less efficiency than wild type MMP-7. It is fully inhibited by EDTA and substrate analogue inhibitors and partially inhibited by N-TIMP-1. This ZBD could be considered the minimum sequence required for catalytic activity, inhibitor docking and substrate recognition.

Keywords

Biology, Molecular; Biology, Cell; Chemistry, Biochemistry

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:9934289