Thyroglobulin Type-1 Domains in Equistatin Inhibit Both Papain-like Cysteine Proteinases and Cathepsin D

Brigita Lenarcic,Vito Turk

doi:10.1074/jbc.274.2.563

Abstract

Equistatin from sea anemone is a protein composed of three thyroglobulin-type 1 domains known to inhibit papain-like cysteine proteinases, papain, and cathepsins B and L. Limited proteolysis was used to dissect equistatin into a first domain, eq d-1, and a combined second and third domain, eq d-2,3. Only the N-terminal domain inhibits papain (Ki = 0.61 nM). Remarkably, equistatin also strongly inhibits cathepsin D with Ki = 0.3 nM but not other aspartic proteinases such as pepsin, chymosin, and HIV-PR. This activity resides on the eq d-2,3 domains (Ki = 0.4 nM). Papain and cathepsin D can be bound and inhibited simultaneously by equistatin at pH 4.5, confirming the physical separation of the two binding sites. Equistatin is the first inhibitor of animal origin known to inhibit cathepsin D. The obtained results demonstrate that the widely distributed thyroglobulin type-1 domains can support a variety of functions.

Highlights

The recently discovered thyroglobulin type-1 domain inhibitors, thyropins, are a group of proteins that have the ability to inhibit both cysteine [1] and a group of as yet uncharacterized cation-dependent proteinases [2]
This has been done by dissecting the molecule by limited proteolysis into folded domain structures, isolating the domains and investigating their inhibitory activities against the cysteine proteinase: papain; a number of aspartic proteinases: cathepsin D, pepsin, chymosin, and HIV-PR; and serine proteinase: trypsin
Equistatin was tested for inhibitory activity against papain, the aspartic proteinases cathepsin D, pepsin, chymosin, and HIV-PR and against serine proteinase, trypsin

Summary

Introduction

The recently discovered thyroglobulin type-1 domain inhibitors, thyropins, are a group of proteins that have the ability to inhibit both cysteine [1] and a group of as yet uncharacterized cation-dependent proteinases [2]. This has been done by dissecting the molecule by limited proteolysis into folded domain structures, isolating the domains and investigating their inhibitory activities against the cysteine proteinase: papain; a number of aspartic proteinases: cathepsin D, pepsin, chymosin, and HIV-PR; and serine proteinase: trypsin. Inhibitory Specificity of Thyroglobulin Type-1 Domains polyvinylidene difluoride membrane and several sheets of blotting paper, was assembled into a blotting apparatus (Biometra) and electroeluted for 1 h at 65 mV in transfer buffer.

Results

Conclusion