Abstract. Mutational studies were previously carried out at the co site in intact cells (Micanovic, R., L. Gerber, J. Berger, K. Kodukula, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA. 87:157-161; Micanovic R., K. Kodukula, L. Gerber, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA: 87:7939-7943) and at the co + 1 and co + 2 sites in a cell-free system (Gerber, L., K. Kodukula, and S. Udenfriend. 1992. J. Biol. Chem. 267:12168-12173) of nascent proteins destined to be processed to a glycosylphosphatidylinositol (GPI)-anchored form. We have now mutated the co + 1 and co + 2 sites in placental alkaline phosphatase (PLAP) cDNA and transfected the wild-type and mutant cDNAs into COS 7 cells. Only glycine at the co + 2 site yielded enzymatically active GPI membrane-anchored PLAP in amounts comparable to the wild type (alanine). Serine was less active and threonine and valine yielded very low but significant activity. By contrast the co + 1 site was promiscuous, with only prollne being inactive. These and the previous studies indicate that the co and ~0 + 2 sites of a nascent protein are key determinants for recognition by COOH-terminal signal transamidase. Comparisons have been made to specific requirements for substitution at the -1, -3 sites of amino terminal signal peptides for recognition by NH2-terminal signal peptidase and the mechanisms of NH2 and COOH-terminal signaling are compared.
We have previously shown that high density lipoprotein is the most abundant protein in the carp plasma and displays bactericidal activity in vitro. Therefore the aim of this study was to analyze the contribution of its principal apolipoproteins, apoA-I and apoA-II, in defense. Both apolipoproteins were isolated by a two step procedure involving affinity and gel filtration chromatography and were shown to display bactericidal and/or bacteriostatic activity in the micromolar range against Gram-positive and Gram-negative bacteria, including some fish pathogens. In addition, a cationic peptide derived from the C-terminal region of carp apoA-I was synthesized and shown to posses antimicrobial activity (EC 50 ¼ 3-6 lM) against Planococcus citreus. This peptide was also able to potentiate the inhibitory effect of lysozyme in a radial diffusion assay at subinhibitory concentrations of both effectors. Finally, limited proteolysis of HDL-associated apoA-I with chymotrypsin in vitro was shown to generate a major truncated fragment, which indicates that apoA-I peptides liberated in vivo through a regulated proteolysis could also be involved in innate immunity.
The distribution of alkaline phosphatase along the carp intestine and also its association to the enterocyte membrane have been studied in order to correlate them with the morphological features and functional specialization of the different intestine portions. The intestine was sectioned in seven segments and from each segment a butanol extract of intestinal alkaline phosphatase (IAP) was prepared. The enzyme activity, when expressed as a function of the mucosa or protein content of each segment showed a clear proximo-distal gradient. In addition, IAP was recovered in the precipitate after centrifugation of the homogenate, along the intestine, indicating that it is membrane associated protein. Also, when brush border membranes (BBM) were isolated, IAP was enriched 10-fold. Butanol extracted IAP from all segments exhibited three bands of activity when separated in PAGE-Triton X-100. The slowest migrating band showed a hydrophobic character as it was retained in a phenyl-Sepharose CL-4B column, whereas the other bands represent hydrophilic IAP found in the flow through of the column. Butanol extracted IAP from BBM rendered only one band with hydrophobic character in PAGE-Triton X-100, which could be converted to hydrophilic forms when incubated with phosphatidyl-inositol phospholipase C. These results clearly demonstrate, that in carp as well as in all other species studied, IAP is anchored to the membrane via a glycosyl-phosphatidylinositol. The high IAP content found in the first segment is consistent with the function of dephosphorylation of nutritional compounds proposed in higher vertebrates. The involvement of IAP in other physiological roles such as lipid absorption or protein internalization cannot be ruled out.
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