Chymotrypsin/trypsin-related serine proteases : A structural, functional and evolutionary analysis

Abstract: Is life possible without proteolytic enzymes? Proteases participate in almost everyaspect of life and they can be found throughout the body and in every cell. Mast cells,together with basophils are, two important effector cells of the immune system. Neutralproteases are the major granule constituents of mast cells and have been also found inbasophils. Besides conferring important biological functions to the mast cells andbasophils, neutral proteases also serve as selective markers for the identification ofdifferent mast cell sub-populations and to distinguish mast cells from basophils. Inaddition, such markers can be used as indicators of allergic inflammation. Studies of apotent IL-4-producing cell population in mice infected with malaria resulted in theidentification of the first basophil specific granule marker in the mouse, MMCP-8.MMCP-8 belongs to a recently identified subfamily of mast cell/basophil serineproteases, clearly distinct from classical chymases and tryptases. To further study thestructure and function of MMCP-8 and its rat homologues, RMCP-8, -9 and 10,recombinant zymogens of these enzymes were expressed in a mammalian expressionsystem. Based on primary amino acid sequences, MMCP-8, RMCP-8, -9 and -10 werefound to have five, three, two and two potential glycosylation sites, respectively. SDS-PAGE analysis of the recombinant proteases strongly indicated that all these potentialsites were used for carbohydrate addition in vivo. In addition, MMCP-8 showed highaffinity for heparin at both pH 5.5, and neutral pH, whereas RMCP-10 bound to heparin only at pH 5.5. Screening for a human homologue to MMCP-8 or another basophil-specific serine protease resulted in the identification of leydin, a novel tyrosine-likeserine protease expressed in the Leydig cells of the testis.Studies of the appearance of the various subfamilies of hematopoietic (and other)serine protease during vertebrate evolution may clarify some of the central questions concerning their biological importance. A screening for novel serine proteases inplatypus, a mammal distantly related to the placentals, led to the isolation of c-DNAclones encoding platypus coagulation factor X, complement factor D and twohematopoietic serine proteases; a neutrophil elastase-like protease and a distantly relatedmember of the T cell granzymes of placental mammals. The isolation of these clonesshows that these four subfamilies of serine protease were already present during earlymammalian evolution. A comparison between aa sequences of these proteases and theircounterparts in placental mammals shows a higher degree of conservation in non-hematopoietic compared to hematopoietic serine proteases.