Cystatin, a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum

Abstract

The protein from chicken egg white that inhibits cysteine proteinases, and has been named 'cystatin', was purified by ovomucin precipitation, affinity chromatography on carboxymethylpapain-Sepharose and chromatofocusing. The final purification step separated two major forms of the protein (pI 6.5 and 5.6), with a total recovery of about 20% from egg white. By use of affinity chromatography and immunodiffusion it was shown that the inhibitor is also present at low concentrations in the serum of male and female chickens. Tryptic peptide maps of the separated forms 1 and 2 of egg-white cystatin were closely similar, and each form had the N-terminal sequence Ser-Glx-Asx. The two forms showed complete immunological identity, and neither contained carbohydrate. Ki values for the inhibition of cysteine proteinases were as follows: papain (less than 1 X 10(-11)M), cathepsin B (8 X 10(-10)M), cathepsin H (about 2 X 10(-8)M) and cathepsin L (about 3 X 10(-12)M). Some other cysteine proteinases, and several non-cysteine proteinases, were found not to be significantly inhibited by cystatin. The inhibition of the exopeptidase dipeptidyl peptidase I by cystatin was confirmed and the Ki found to be 2 X 10(-10)M. Inhibitor complexes with active cysteine proteinases and the inactive derivatives formed by treatment with iodoacetate, E-64 [L-trans-epoxysuccinylleucylamido(4-guanidino)butane] and benzyloxycarbonylphenylalanylalanyldiazomethane were demonstrated by isoelectric focusing and cation-exchange chromatography. The complexes dissociated in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with or without reduction) with no sign of fragmentation of the inhibitor. Cystatin was found not to contain a free thiol group, and there was no indication that disulphide exchange plays any part in the mechanism of inhibition.

The cysteine proteinase inhibitor chicken cystatin is a phosphoprotein

“…Thus, it is quite uncertain as to whether the relative amounts of forms 1 and 2 found after the purification procedures reflect the in vivo situation. Furthermore, since chicken cystatin is not present exclusively in egg white but has also been detected in the serum and in muscle cells [4,24], variable amounts of the two isoelectric forms may be present in different tissues.…”

“…By ion-exchange chromatography chicken cystatin can be resolved into two immunologically identical forms with pI values of 6.5 and 5.6 [4], designated as forms A and B [5] or 1 and 2 [4], respectively. The structural differences responsible for the occurrence of two isoelectric forms are a matter of controversy.…”

“…Endoproteinases Glu-C and Asp-N were purchased from Boehringer. [4], fragments were separated on a Macherey-Nagel ET 250/8/4 Nucleosil 300-10 C-18 HPLC column in an elution gradient from 0 (solvent A) to 80% acetonitrile (solvent B) in 0.1% TFA in 160 min.…”

Functional expression of recombinant human stefin A in mammalian and bacterial cells

“…The relative increase in fluorescence was measured at an excitation wavelength of 370 nm and emission wavelength of 460 nm. Inhibition constant, K i , was determined according to the method of Nicklin and Barrett (26) …”

Mechanism of inhibition of papain by chicken egg white cystatin

“…It forms a tight, reversible 1 : 1 complex with most known cysteine proteinases [3]. Chicken egg white cystatin has been reported to occur in two major isoelectric forms, form 1 (pI = 6.5) and form 2 (pl = 5.6), which can be separated by ion exchange chromatography [4,5]. Both forms represent the full-length inhibitors starting with Ser (Ser-form) and comprising 116 amino acid residues Correspondence address: W. Machleidt, Institut ffir Physiologische Chemie, Physikalische Biochemie und Zellbiologie tier Universit~it Miinchen, Goethestrafle 33, D-8000 Miinchen 2, FRG…”