Human Cystatin A Is Inactivated by Engineered Truncation. The NH2-Terminal Region of the Cysteine Proteinase Inhibitor Is Essential for Expression of Its Inhibitory Activity

Abstract

A series of NH2-terminal truncated forms of human cysteine proteinase inhibitor, cystatin A, was prepared by genetic engineering using Escherichia coli harboring mutated genes. Each variant of cystatin A was efficiently expressed as a fused protein with porcine adenylate kinase and released by CNBr degradation after exchange of the sole inner Met to Leu. The mutant cystatin A lacking an amino-terminal Met residue (called standard variant starting from Ile2, CystA2-98(M65L) showed the same inhibitory activity as authentic one isolated from human epidermis. Two-residue truncation scarcely influenced the activity, but further truncations deleting Pro3 and beyond conservative Gly4 and Gly5 caused a remarkable decrease of their inhibitory activity. But little effect was observed by a substitution of Pro3 with Leu. The loss of the activity by amino-terminal truncation was compensated slightly by engineered substitution of Gly75 with His on a second loop. In the two-dimensional 15N-1H HSQC NMR spectrum, four-residue truncation was found to cause changes in the chemical shifts of Val47 and Val48, which locate on a first loop and consist of a conservative QVVAG sequence. Furthermore, the truncation led to a change in fluorescence spectroscopic behavior of Trp75, which was introduced as a probe on the second loop. Fluorescence intensity of the Trp of the truncated (5-98) form was more affected by heating than the active standard variant. Conversely, fluorescence of Trp75 in 2-98 form was more quenched by acrylamide than the 5-98 variant. Thus, the amino-terminal region of cystatin A is essential for the expression of its inhibitory activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Leukocystatin, A New Class II Cystatin Expressed Selectively by Hematopoietic Cells

“…The FLAGtagged material was isolated as soluble material from the periplasm; the GST fusion, however, was primarily recovered as inclusion bodies which had to be refolded. Although the Class I cystatin A requires refolding after expression in E. coli (21,22), no other Class II cystatin is insoluble when expressed in E. coli, even when expressed as the GST fusion (20). The mature protein was isolated following thrombin cleavage of the GST moiety or enterokinase cleavage of the FLAG tag.…”

“…Although some of these products require refolding, the FLAG-tagged material was soluble, similar to other Class II members expressed in E. coli, including those used for comparison in Table II (16,17). Although the Class I cystatin A requires refolding following overexpression in E. coli, this was shown to have no adverse effect on activity (21,22). We further controlled for any effects that refolding may have on activity by examining denatured/ renatured chicken egg white cystatin and found no difference in activity following this step.…”

Bricks and mortar of the epidermal barrier

“…Cystatin A is a substrate for epidermal TGases and has been identified as a minor cross-linked component of CEs (Zettergren et a l., 1984;Takahashi et al, 1992;Steinert and Marekov, 1997). The protein is a known cysteine protease inhibitor (Jarvinen et al ., 1987;Takahashi et al, 1994;Shibuya et al, 1995) and it has been suggested that this feature might be relevant for the bacteriostatic properties of the skin (Takahashi e t al., 1994).…”

Structural organization of cornified cell envelopes and alterations in inherited skin disorders

“…Cystatin A is a 12 kDa, lysine-rich, cysteine proteinase inhibitor (79,80 , is essential for the proteinase inhibitor activity (83). An amino acid residue involved in the dipeptide crosslink is Lys 46 (15).…”