EVOLUTION OF PARALOGOUS AVIAN INHIBITORS OF SERINE PROTEINASES

WARREN CHAUNCEY BOGARD, Purdue University

Abstract

Japanese quail ovomucoid was shown to exist in two polymorphic forms. One has serine, the other glycine at position 162 in the primary structure. The tryptic peptide corresponding to position 160-164 was purified from ovomucoids isolated from egg whites of eggs laid by eleven different hens and subjected to amino acid analysis. The quantitative distribution of serine and glycine in this pentapeptide is consistent with the interpretation that the ovomucoid gene exists in two codominant allelic forms at one locus. Even though the gene product is apparently expressed only in the female, these results indicate that the ovomucoid structural gene is transmitted as a simple Mendelian character which is neither sexlinked nor shows dominance. Thus, non-Mendelian modes of inheritance cannot account for the unusual evolutionary behavior of ovomucoid {Kato et al. (1978) in FEBS 11th Meeting: Regulatory Proteolytic Enzymes and Their Inhibitors (Magnusson et al., Eds.) Vol. 47, pp 197-206, Pergamon Press, Oxford}. Intact third domains (position 131-186) isolated from the two allelic forms of ovomucoid interact with bovine (beta)-trypsin in a similar but not identical manner; the complex with the glycine form dissociates three times more rapidly. Evidence is presented which suggests that glycine is the ancestral residue at position 162; yet, the serine form is the more frequent phenotype. Turkey pancreatic secretory trypsin inhibitor (PSTI) was isolated ((TURN)30 (mu)g per animal) from non-autolyzed turkey pancreatic tissue by a combination of gel exclusion chromatography and affinity chromatography on insolubilized anhydrotrypsin. The inhibitor was characterized by amino acid analysis and by sequencer analysis. The complete primary structure is: 5 10 15 1 Asn Ala Glu Pro Asp Gly Ala Ala Gly Gln Gly Thr Glu Ala Ala 16 Cys Gly Asn Tyr Asp Val Arg Lys Gly Cys Thr Lys Asn Phe Asp 31 Pro Ile Cys Gly Thr Asp Asp Val Leu Tyr Ser Asn Glu Cys Leu 46 Leu Cys Val Gln Asn Met Gln Arg His Thr Asn Val Arg Ile Lys 61 Asn Arg Gly Lys Cys Gln Glu Pro Ser Pro Arg (Ser). As isolated, the inhibitor is a nearly equimolar mixture of two forms: PSTYKY which was the sequence indicated above and PSTKYA which has an identical sequence minus the amino-terminal dipeptide Asn-Ala. PSTKYA does not appear to be an isolation artifact. The inhibitor is clearly homologous and probably orthologous to the mammalian pancreatic secretory trypsin inhibitors as is shown by a comparison of the turkey and human {Bartelt et al. (1977) Arch. Biochem. Biophys. 179, 189-199} PSTI reactive site region sequences (symbolized PSTKY and PSHUM). PSTKY ...Gly-Cys('25)-Thr-Lys-Asn-Phe-Asp('30)-Pro-Ile-Cys-Gly... PSHUM ...Gly-Cys('16)-Thr-Lys-Ile-Tyr-Asn('21)-Pro-Val-Cys-Gly... There are however significant differences from the mammalian inhibitors. These include an appreciably longer amino terminus, a dipeptide insertion in the region between the first and second cysteines, a carboxylterminal extension beyond the sixth cysteine, and several non-conservative amino acid replacements. The turkey pancreatic secretory trypsin inhibitor is not one of the three Kazal-type domains of ovomucoid (by inspection) nor any of the seven Kazal-type domains of turkey ovoinhibitor (by comparison to chicken ovoinhibitor and other indirect evidence). Thus, the turkey genome codes for at least eleven paralogous Kazal-type inhibitor domains.

Degree

Ph.D.

Subject Area

Biochemistry

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