Expression and characterization of human tissue kallikrein variants.
Human tissue kallikrein is a serine protease implicated in the pathology of various inflammatory disorders. As one of the two principal enzymes that generate proinflammatory kinin peptides in vivo, tissue kallikrein represents an attractive target for therapeutic intervention in diseases such as asthma, pancreatitis, and rheumatoid arthritis. Three distinct human tissue kallikrein variants, differing in one or two amino acid substitutions, are predicted to exist based on genomic or cDNA nucleotide sequences derived from different tissues. The effects of these substitutions on the biochemical properties of tissue kallikrein are unknown but could, in principle, confer tissue-specific functions on the enzyme or affect the clinical utility of specific kallikrein inhibitors. All three variants, as well as a deglycosylated derivative, were expressed in high yield as recombinant proteins in Pichia pastoris. The recombinant kallikrein variants and natural urinary kallikrein all hydrolyzed synthetic peptides with similar specificity and efficiency and released kallidin from kininogen at comparable rates. Similarly, no significant differences were observed in the interactions between kallikrein variants and protein inhibitors such as SBTI, alpha1-PI, and aprotinin. We conclude that the known tissue kallikrein variants represent allelic variants and are not likely to have tissue-specific activity related to the amino acid substitutions.
Chan
H
H
Department of Molecular Biology, Axys Pharmaceuticals Corporation, 180 Kimball Way, South San Francisco, California 94080, USA. hedy_chan@axys.com
Springman
E B
EB
Clark
J M
JM
D000595:Amino Acid Sequence; D007611:Aprotinin / Q000494:pharmacology; D001483:Base Sequence; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D006801:Humans; D007610:Kallikreins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D007704:Kininogens / Q000378:metabolism; D007705:Kinins / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010179:Pancreas / Q000201:enzymology*; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012469:Salivary Glands / Q000201:enzymology*; D014361:Trypsin Inhibitors / Q000494:pharmacology*; D000515:alpha 1-Antitrypsin / Q000494:pharmacology
D015853:Cysteine Proteinase Inhibitors; D017931:DNA Primers; D007704:Kininogens; D007705:Kinins; D009842:Oligopeptides; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D000515:alpha 1-Antitrypsin; D007611:Aprotinin; D007610:Kallikreins