Contribution of the C-terminal domain of metalloproteinases to binding by tissue inhibitor of metalloproteinases: C-terminal truncated stromelysin and matrilysin exhibit equally compromised binding affinities as compared to full-length stromelysin

Vijaykumar M. Baragi, Catherine J. Fliszar, Mary Carol Conroy, Qizhuang Ye, J. Michael Shipley, Howard G. Welgus

Research output: Contribution to journalArticle

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Abstract

In this study, we have used high resolution gel-filtration chromatography and measurements of Ki to compare the capacity of full-length native stromelysin, C-terminal truncated stromelysin (Phe100-Pro273), and matrilysin (the only metalloproteinase spontaneously lacking a C-terminal hemopexin-like domain) to bind to the tissue inhibitor of metalloproteinases (TIMP). While prostromelysin failed to bind TIMP, active stromelysin bound to the inhibitor avidly, exhibiting an affinity for TIMP (Ki = 8.3 × 10-10 M) essentially identical to that of active interstitial collagenase as determined by competition experiments. C-terminal truncated stromelysin also formed a higher Mr complex with TIMP which survived gel filtration. However, when truncated stromelysin was forced to compete with its full-length parent molecule for limiting amounts of TIMP, the full-length enzyme preferentially bound to the inhibitor. Indeed, binding studies indicated a Ki of 5.95 × 10-9 M for the truncated variant's interaction with TEMP, only 14% as tight as that of full-length stromelysin. We also examined the interaction between TIMP and matrilysin, the only metalloproteinase which naturally lacks a C-terminal domain. Promatrilysin failed to bind the inhibitor. However, active matrilysin readily bound TIMP, forming a complex that resisted separation by gel filtration. When active matrilysin was forced to compete with truncated stromelysin for limiting amounts of TIMP, both enzymes appeared to complex the inhibitor with nearly equivalent efficacy. Indeed, active matrilysin exhibited a Ki for TIMP of 4.5 × 10-9 M, essentially identical to that of truncated stromelysin. These data indicate that, as is true for collagenase, the C-terminal domain of stromelysin contributes significantly to its capacity to bind the physiologic inhibitor, TIMP. Furthermore, since stromelysin readily processes in vitro to a C-terminal truncated form, this enzyme species, as well as the full-length metalloproteinase matrilysin, may resist inhibition by TIMP in areas of active inflammation in vivo.

Original languageEnglish (US)
Pages (from-to)12692-12697
Number of pages6
JournalJournal of Biological Chemistry
Volume269
Issue number17
StatePublished - Apr 29 1994
Externally publishedYes

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Matrix Metalloproteinase 7
Tissue Inhibitor of Metalloproteinases
Matrix Metalloproteinase 3
Metalloproteases
Gel Chromatography
Gels
Enzymes
Hemopexin
Matrix Metalloproteinase 1
Collagenases
Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Contribution of the C-terminal domain of metalloproteinases to binding by tissue inhibitor of metalloproteinases : C-terminal truncated stromelysin and matrilysin exhibit equally compromised binding affinities as compared to full-length stromelysin. / Baragi, Vijaykumar M.; Fliszar, Catherine J.; Conroy, Mary Carol; Ye, Qizhuang; Shipley, J. Michael; Welgus, Howard G.

In: Journal of Biological Chemistry, Vol. 269, No. 17, 29.04.1994, p. 12692-12697.

Research output: Contribution to journalArticle

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title = "Contribution of the C-terminal domain of metalloproteinases to binding by tissue inhibitor of metalloproteinases: C-terminal truncated stromelysin and matrilysin exhibit equally compromised binding affinities as compared to full-length stromelysin",
abstract = "In this study, we have used high resolution gel-filtration chromatography and measurements of Ki to compare the capacity of full-length native stromelysin, C-terminal truncated stromelysin (Phe100-Pro273), and matrilysin (the only metalloproteinase spontaneously lacking a C-terminal hemopexin-like domain) to bind to the tissue inhibitor of metalloproteinases (TIMP). While prostromelysin failed to bind TIMP, active stromelysin bound to the inhibitor avidly, exhibiting an affinity for TIMP (Ki = 8.3 × 10-10 M) essentially identical to that of active interstitial collagenase as determined by competition experiments. C-terminal truncated stromelysin also formed a higher Mr complex with TIMP which survived gel filtration. However, when truncated stromelysin was forced to compete with its full-length parent molecule for limiting amounts of TIMP, the full-length enzyme preferentially bound to the inhibitor. Indeed, binding studies indicated a Ki of 5.95 × 10-9 M for the truncated variant's interaction with TEMP, only 14{\%} as tight as that of full-length stromelysin. We also examined the interaction between TIMP and matrilysin, the only metalloproteinase which naturally lacks a C-terminal domain. Promatrilysin failed to bind the inhibitor. However, active matrilysin readily bound TIMP, forming a complex that resisted separation by gel filtration. When active matrilysin was forced to compete with truncated stromelysin for limiting amounts of TIMP, both enzymes appeared to complex the inhibitor with nearly equivalent efficacy. Indeed, active matrilysin exhibited a Ki for TIMP of 4.5 × 10-9 M, essentially identical to that of truncated stromelysin. These data indicate that, as is true for collagenase, the C-terminal domain of stromelysin contributes significantly to its capacity to bind the physiologic inhibitor, TIMP. Furthermore, since stromelysin readily processes in vitro to a C-terminal truncated form, this enzyme species, as well as the full-length metalloproteinase matrilysin, may resist inhibition by TIMP in areas of active inflammation in vivo.",
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T2 - C-terminal truncated stromelysin and matrilysin exhibit equally compromised binding affinities as compared to full-length stromelysin

AU - Baragi, Vijaykumar M.

AU - Fliszar, Catherine J.

AU - Conroy, Mary Carol

AU - Ye, Qizhuang

AU - Shipley, J. Michael

AU - Welgus, Howard G.

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