The cysteine proteinase inhibitor chicken cystatin is a phosphoprotein

Abstract

Peptide maps obtained by reversed-phase HPLC of tryptic digests of isoelectric form 1 (pl= 6.5) and 2 (pl = 5.6) of chicken egg white cystatin revealed that the difference was located only in a single peptide (residues Ser-74-Lys-91). Ser-80 of cystatin 2 was subsequently identified as being modified by phosphorylation. Moreover, alkaline phosphatase treatment of a mixture of native cystatin forms. 1 and 2 was shown by ion-exchange chromatography to cause the disappearance ofisoetectric form 2 with a concomitant increase in form 1. Thus, the existence of two isoelectric forms of chicken cystatin is due to the phosphorylated form 2 and non-phosphorylated form 1.

A Virus Essential for Insect Host-Parasite Interactions Encodes Cystatins

“…1B) (49). Cystatin 1 possesses a putative tyrosine phosphorylation site at position 121 to 127, and cystatins 3 and 2 contain one and two potential Asn-linked glycosylation sites, respectively (Prosite, Expasy; http://www .expasy.org/prosite/), in accord with published reports suggesting that at least some cystatins are phosphorylated (40) and glycosylated (20).…”

Purification and characterization of a chicken egg white cystatin variant expressed in an Escherichia coli pIN‐III‐ompA system

“…Our two-step purification procedure led to a homogeneous mini-protein of high purity, with inhibition constants which are very similar to those of native cystatin. Although the measurable specific inhibitory activity of 50% obtained for chicken AEF-[SI -+ M, M29 -+I, M89 -+ Llcystatin is rather low, it is comparable to the values detected for the highly purified full-length non-phosphorylated form of native chicken cystatin (62 -75% inhibitory activity) isolated by Laber et al [8,121. Inhibitory material could not be separated from non-inhibitory material, i. e. both were indistinguishable by native PAGE, SDS/PAGE, HPLC and NMR spectroscopy.…”

“…Isolation of pure native chicken cystatin is very tedious [8,12,131. Our two-step purification procedure led to a homogeneous mini-protein of high purity, with inhibition constants which are very similar to those of native cystatin.…”

“…Papain (Sigma), repurified on HgSepharose, was kindly provided by B. Laber, Munich, and actinidin and cathepsin B were generous gifts from V. Turk, Ljubljana. Natural highly purified chicken cystatin was native, full-length and in the non-phosphorylated form [8,121. The inhibition constants with actinidin were determined in continuous assay systems using 0.02 -0.1 mM Z-Phe-Arg-NHMec, 0.6 -6.0 nM actinidin and 5 -150 nM inhibitor.…”

Cystatin M suppresses the malignant phenotype of human MDA-MB-435S cells

“…(n ¼ 8) and compared statistically using one-way ANOVA. Adhesion measurements were normalized to that of the mock clone to clearly identify the differences in tumor cell adhesion undergo N-or O-glycosylation, Ser/Thr-phosphorylation, dimerization, or to bind calcium ions (Bell et al, 1989;Laber et al, 1989;Ekiel et al, 1997;Merz et al, 1997;Taupin et al, 2000). Further studies are required to determine which of these modifications occur in cystatin M and are important for its antiproliferative function.…”