Cloning and expression of ostrich trypsinogen: an avian trypsin with a highly sensitive autolysis site.
One of ostrich (Struthio camelus) trypsinogen genes was cloned from pancreatic cDNA. Its amino acid sequence compared to known trypsin sequences from other species shows high identity and suggests that it is a member of the phylogenetically anionic trypsinogen I subfamily. After cytoplasmic over expression in Escherichia coli and renaturation, the activation properties of ostrich trypsinogen were studied and compared to those of human trypsinogen 1 (also called as human cationic trypsinogen). Ostrich trypsinogen undergoes bovine enterokinase activation and autoactivation much faster than human trypsinogen 1 and exhibits on a synthetic substrate a somewhat higher enzymatic activity than the latter one. The most interesting property of ostrich trypsin is its relatively fast autolysis that can be explained via a mechanism different from the common mechanism for rat and human 1 trypsins. The latter proteases have a site, Arg117-Val118, where the autolysis starts and then goes on in a zipper-like fashion. This is absent from ostrich trypsin. Instead it has a couple of cleavage sites within regions 67-98, including two unusual ones, Arg76-Glu77 and Arg83-Ser84. These appear to be hydrolysed fast in a non-consecutive manner. Such an autolysis mechanism could not be inhibited by a single-site mutation which in humans is proposed to lead to pancreatitis.
Szenthe
Borbála
B
Department of Biochemistry, Eötvös Lóránd University, Pázmány Péter sétány 1/C, H-1117 Budapest, Hungary.
Frost
Carminita
C
Szilágyi
László
L
Patthy
András
A
Naudé
Ryno
R
Gráf
László
L
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D004765:Enteropeptidase / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020310:Struthioniformes / Q000378:metabolism*; D014362:Trypsinogen / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism*
D014362:Trypsinogen; D004765:Enteropeptidase