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Doctoral thesis, 1999

Characterisation of two endogenous mammalian cysteine proteinase inhibitors, bovine cystatin C and human cystatin A

Olsson, Sigrid-Lisa

Abstract

The binding of human cystatin A to papain-like proteinases was quantified with a recombinant inhibitor, expressed in E.coli. The interaction with papain and cathepsin L was strong, with K, values of 10'n-10'13 M, and rapid, with values of 3-5106 M’1*1. The binding to papain was consistent with a one-step reaction, occurring without detectable conformational changes ofeitherproteinase orinhibitor.

Hie cDNA sequence of bovine cystatin C and analyses of the inhibitor isolated from cerebrospinal fluid showed that bovine cystatin C is synthesised as a preprotein with a signal peptide of 30 residues preceding the mature protein with 118 residues. In particular, the inhibitor has an N-terminal region similar to that of other family II cystatins.

Recombinant bovine cystatin C with a complete N-terminal region was characterised. The general properties, as well as the affinity and kinetics of inhibition of papain and cathepsins B, H and L, were comparable with those of human cystatin C. However, some differences between the bovine and human inhibitors were observed. Most importantly, bovine cystatin C bound to cathepsin L with a four-fold higher association rate constant than the human inhibitor. The full-length bovine cystatin C bound appreciably more tightly to proteinases than the shorter forms characterised previously. Digestion with elastase indicated that these forms had arisen by cleavage of a full-length inhibitor.

In-situ hybridisation with digoxigenin-labelled cRNA probes demonstrated that bovine cystatin C mRNA was heavily concentrated in the epithelial cells ofthe choroid plexus, in single cells speckled in lymphoid tissue and in Sertoli cells. Cystatin C mRNA was also present in occasional neurons and glial cells throughout the cerebrum and the cerebellum. In the submandibular gland, specific mRNA was found mainly in striated intralobular and interlobular ducts. The expression of cystatin C in brain tissue is of particular interest, as the inhibitor is involved in certain neurological diseases.

Keywords

cysteine proteinase; cysteine proteinase inhibitor; cystatins; bovine; cerebrospinal fluid; in-situ hybridisation; enzyme kinetics; papain; cathepsins

Published in

Acta Universitatis Agriculturae Sueciae. Veterinaria
1999, number: 51
ISBN: 91-576-5423-9
Publisher: Swedish University of Agricultural Sciences

Authors' information

Olsson, Sigrid-Lisa
Swedish University of Agricultural Sciences, Department of Veterinary Medical Chemistry

UKÄ Subject classification

Pathobiology
Medicinal Chemistry

URI (permanent link to this page)

https://res.slu.se/id/publ/117454