Purification and molecular cloning of chymase from human tonsils

Abstract

A chymotrypsin‐like protease was purified to homogeneity from human tonsils by a series of Chromatographie procedures. The purified enzyme gave a single protein band with an apparent molecular mass of 30 kDa on SDS‐PAGE. The sequence of the first 21 amino acids at the N‐terminus of the enzyme was determined. A cDNA for the enzyme was cloned by PCR amplification from extracted tonsillar mRNA using a supposed N‐terminal oligonucleotide primer and a conserved C‐terminal primer of the chymase family. The deduced amino acid sequence of the isolated clone was identical to that of human chymase in connective tissue‐type mast cells from heart except for a Ser instead of a Cys at the N‐terminal 7th position.

Two distinct forms of human mast cell chymase

“…This discrepancy may be accounted for by one or more of the following differences in chromatographic conditions: matrix (TSK heparin versus heparin-agarose), salt (KCl versus NaCl), pH (8.0 versus 6.8) and Triton X-100 (gradient of 0.0 to 0.1 % versus a constant 0.03%). Chymase from human tonsil tissue has been reported to be eluted from a heparin-agarose column at 1.2 M NaCl [25], the same as chymase B in the present studies.…”

“…Only one form has been purified from skin [24], heart [4] and tonsil [25] tissue. All these isolates appear to have identical amino acid sequences [25Ϫ27], and an identical sequence is also predicted from cDNA from the uterus [28].…”

Induction of mast cell accumulation by chymase via an enzymatic activity‐ and intercellular adhesion molecule‐1‐dependent mechanism

“…Since chymase belongs to the chymotrypsin family of protease (Sukenaga et al, ) and chymase‐induced mast cell accumulation may depend on its enzymatic activity, we employed the common chymotryptic enzymes α 1 ‐chymotrypsin (Niemann, ) and elastase as positive and negative controls, respectively, in the present study. The results showed that α 1 ‐chymotrypsin also induced a dose‐dependent increase in mast cell accumulation at 6 h (Figure E) and 16 h (Figure F) following injection.…”

Production of crystallizable human chymase from a Bacillus subtilis system

“…For example, human chymase activates angiotensin I to angiotensin II by cleavage of a Phe–His bond, but does not degrade angiotensin II by clipping a Tyr–Ile bond, as do chymotrypsin, and chymases from other species [9]. To date, only one chymase gene has been identified in humans [13], although a Cys‐to‐Ser variation at position seven of the mature enzyme has been reported [14]. This is the first report of crystallization of human chymase.…”