Purification and characterization of novel kininogens from spotted wolffish and Atlantic cod.

Kininogens are multifunctional proteins found so far mainly in mammals. They carry vasoactive kinins as well as participate in defense, blood coagulation and the acute phase response. In this study, novel kininogens were isolated from Atlantic cod (Gadus morhua L.) and spotted wolffish(Anarhichas minor) by papain-affinity chromatography. The molecular mass of cod kininogen determined by MALDI-TOF mass spectrometry to be 51.0 kDa and it had pI values of 3.6, 3.9 and 4.4. The molecular mass of wolffish kininogen was 45.8 kDa and it had pI values of 4.1, 4.3, 4.35 and 4.4. Partial amino-acid sequences determined from both kininogens showed clear homology with previously determined kininogen sequences. Both kininogens were found to inhibit cysteine proteinases like papain and ficin but they had no effect on trypsin, a serine proteinase. Wolffish kininogen carried alpha2,3-sialylated biantennary and triantennary N-glycans with extensive sialic acid O-acetylation. Cod kininogen carried similar glycan structures but about 1/3 of its glycans carried sulfate at their N-acetylglucosamine units.

Immunolocalization of cysteine proteinases (cathepsins) and cysteine proteinase inhibitors (salarin and salmon kininogen) in Atlantic salmon, Salmo salar.

Tissue localization of cysteine proteinases (cathepsins) and their inhibitors (salarin, salmon kininogen) was performed in tissues of the Atlantic salmon. In skin, both epidermis and dermis were strongly stained by antisera against salarin and salmon kininogen. In epidermis the intercellular space seemed to be heavily stained (salarin). In kidney, the inhibitors were mainly localized to the interstitial capillaries. Also, some epithelial cells of the tubules (salarin) and some cells of the interstitium were stained. Mostly, the staining had a diffuse cytoplasmic localization. In the liver some hepatocytes were strongly positive for salarin and salmon kininogen. Purified fish cysteine proteinase inhibitors were not found to inhibit the growth of fish pathogenic bacteria and viruses. In the trunk kidney cathepsins B and L were localized in epithelial cells of the tubules (proximal part) and in cells of the interstitium. Mostly, the staining showed a prominent lysosomal localization. In head kidney large macrophage-like cells were positively stained for cathepsin B. The staining was localized to granula/vacuoles in the cytoplasm. In the liver, some hepatocytes were strongly stained and some were less strongly positive for cathepsin B and L. Mostly, the hepatocytes showed lysosomal staining. Cathepsin L was found in some big macrophage-like cells in the spleen. Mucosal epithelial cells of the esophagus and intestine seemed to be strongly stained for cathepsin B and L. The results show that cathepsins and their inhibitors are specifically and widely distributed in the Atlantic salmon skin indicating that they perform some biologically important and specific but so far unknown functions.