Porcine kallikrein-4 activation, glycosylation, activity, and expression in prokaryotic and eukaryotic hosts.
Kallikrein-4 (KLK4) is a serine proteinase believed to be important in the normal development of dental enamel. We isolated native KLK4 from developing pig enamel and expressed four recombinant forms. Pig KLK4 was expressed in bacteria with and without the propeptide, and in two eukaryotic systems. Recombinant pig KLK4 was secreted as a zymogen by '293' cells and purified. The proKLK4 was activated in vitro by thermolysin and recombinant pig enamelysin, but not by native KLK4. These results were confirmed using a fluorescent peptide analog of the KLK4 propeptide-enzyme junction. Native KLK4 appears as a doublet at 37 kDa and 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Removal of N-linked oligosaccharides by digestion with deglycosidase-F reduced the doublet to a single band at approximately 28 kDa, demonstrating that the active enzyme is glycosylated, and that the 37 kDa and 34 kDa forms differ only in their number of glycosylations. Deglycosylation was also associated with a loss of proteolytic activity. We digested recombinant pig amelogenin with native KLK4 and characterized the cleavage products by N-terminal sequencing and mass spectrometry. Eleven cleavage sites in the amelogenin protein were identified, demonstrating that KLK4 degrades amelogenin and is likely to participate in the degradation of enamel proteins in vivo.
Ryu
Okhee
O
University of Texas School of Dentistry, Health Science Center at San Antonio, Department of Pediatric Dentistry, San Antonio, Texas, USA.
Hu
Jan C C
JC
Yamakoshi
Yasuo
Y
Villemain
Jana L
JL
Cao
Xiaohang
X
Zhang
Chuhua
C
Bartlett
John D
JD
Simmer
James P
JP
D053523:Amelogenin; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D003746:Dental Enamel Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D007610:Kallikreins / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D053519:Matrix Metalloproteinase 20; D020782:Matrix Metalloproteinases / Q000494:pharmacology; D009844:Oligosaccharides / Q000032:analysis; D011994:Recombinant Proteins; D013552:Swine; D013820:Thermolysin / Q000494:pharmacology
D053523:Amelogenin; D003746:Dental Enamel Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D007610:Kallikreins; C120801:kallikrein 4; D053519:Matrix Metalloproteinase 20; D020782:Matrix Metalloproteinases; D013820:Thermolysin