Functional evolution within a protein superfamily.

The ability to predict and characterize distributions of reactivities over families and even superfamilies of proteins opens the door to an array of analyses regarding functional evolution. In this article, insights into functional evolution in the Kazal inhibitor superfamily are gained by analyzing and comparing predicted association free energy distributions against six serine proteinases, over a number of groups of inhibitors: all possible Kazal inhibitors, natural avian ovomucoid first and third domains, and sets of Kazal inhibitors with statistically weighted combinations of residues. The results indicate that, despite the great hypervariability of residues in the 10 proteinase-binding positions, avian ovomucoid third domains evolved to inhibit enzymes similar to the six enzymes selected, whereas the orthologous first domains are not inhibitors of these enzymes on purpose. Hypervariability arises because of similarity in energetic contribution from multiple residue types; conservation is in terms of functionality, with "good" residues, which make positive or less deleterious contributions to the binding, selected more frequently, and yielding overall the same distributional characteristics. Further analysis of the distributions indicates that while nature did optimize inhibitor strength, the objective may not have been the strongest possible inhibitor against one enzyme but rather an inhibitor that is relatively strong against a number of enzymes.

Structural and functional study of an Anemonia elastase inhibitor, a "nonclassical" Kazal-type inhibitor from Anemonia sulcata.

Anemonia elastase inhibitor (AEI) is a "nonclassical" Kazal-type elastase inhibitor from Anemonia sulcata. Unlike many nonclassical inhibitors, AEI does not have a cystine-stabilized alpha-helical (CSH) motif in the sequence. We chemically synthesized AEI and determined its three-dimensional solution structure by two-dimensional NMR spectroscopy. The resulting structure of AEI was characterized by a central alpha-helix and a three-stranded antiparallel beta-sheet of a typical Kazal-type inhibitor such as silver pheasant ovomucoid third domain (OMSVP3), even though the first and fifth half-cystine residues forming a disulfide bond in AEI are shifted both toward the C-terminus in comparison with those of OMSVP3. Synthesized AEI exhibited unexpected strong inhibition toward Streptomyces griseus protease B (SGPB). Our previous study [Hemmi, H., et al. (2003) Biochemistry 42, 2524-2534] demonstrated that the site-specific introduction of the engineered disulfide bond into the OMSVP3 molecule to form the CSH motif could produce an inhibitor with a narrower specificity. Thus, the CSH motif-containing derivative of AEI (AEI analogue) was chemically synthesized when a Cys(4)-Cys(34) bond was changed to a Cys(6)-Cys(31) bond. The AEI analogue scarcely inhibited porcine pancreatic elastase (PPE), even though it exhibited almost the same potent inhibitory activity toward SGPB. For the molecular scaffold, essentially no structural difference was detected between the two, but the N-terminal loop from Pro(5) to Ile(7) near the putative reactive site (Met(10)-Gln(11)) in the analogue moved by 3.7 A toward the central helix to form the introduced Cys(6)-Cys(31) bond. Such a conformational change in the restricted region correlates with the specificity change of the inhibitor.

Surfactants enhance the tight-junction permeability of food allergens in human intestinal epithelial Caco-2 cells.

BACKGROUND: Food additives are responsible for certain allergic types of symptoms. Here Caco-2 cell monolayers were used as a model of the intestinal epithelium for the study of the effect of a food grade surfactant. We determined whether or not the presence of a surfactant enhances the transportation of food allergens across human intestinal epithelial Caco-2 cells. METHODS: This study investigated sucrose monoester fatty acids, which are a food grade surfactant. As an in vitro model of human epithelial cells, Caco-2 cells were grown in monolayers and exposed to different doses of the surfactant in conjunction with ovomucoid, a major egg white allergen. The integrity of the monolayer was assessed by measuring transepithelial electrical resistance (TEER). The permeability of tight junctions and transport of the antigen were studied. RESULTS: TEER correlated with the permeability of tight junctions. TEER significantly decreased upon exposure to a surfactant, indicating an increase in ovomucoid permeability without degradation. The surfactant induced shortening in microvilli, actin disbandment and structural separation of tight junctions. The results indicate that food grade surfactants can increase the paracellular uptake of food allergens.

Egg ovomucin attenuates hypercholesterolemia in rats and inhibits cholesterol absorption in Caco-2 cells.

This experiment was designed to evaluate the effect of casein or ovomucin (OV) on the micellar solubility of cholesterol and the taurocholate binding capacity in vitro. We also evaluated the effects of casein or OV on cholesterol metabolism in rats and Caco-2 cells. OV had a significantly greater bile acid-binding capacity than that of casein in vitro. Micellar cholesterol solubility in vitro was significantly lower in the presence of OV compared to casein. The cholesterol micelles containing OV significantly suppressed cholesterol uptake by Caco-2 cells compared to the cholesterol micelles containing casein. Consistent with these in vitro findings, OV-feeding significantly increased the fecal excretion of bile acids or cholesterol compared with casein-feeding. Serum total cholesterol was significantly lower in rats fed OV than in those fed casein. The concentrations of total lipids in liver were significantly lower in the OV-fed group compared with the casein group. These results suggest that the suppression of cholesterol absorption by direct interaction between cholesterol mixed micelles and OV in the jejunal epithelia is part of the mechanism underlying the hypocholesterolemic action of OV. OV may also inhibit the reabsorption of bile acids in the ileum, thus lowering the serum cholesterol level.

Secretory production of chicken ovomucoid domain 3 by Escherichia coli and alteration of inhibitory specificity toward proteases by substitution of the P1 site residue.

Ovomucoids are commonly present in bird egg white and exhibit inhibitory activity toward various serine proteases. To investigate the structure-function relationship of ovomucoid domain 3, we established a secretory expression system for the chicken ovomucoid domain 3 (OMCHI3)-encoding gene in Escherichia coli by ligating it downstream from the tac promoter and signal peptide of E. coli alkaline phosphatase. E. coli JM105 was transformed with the resulting plasmid and induced with 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). The mature OMCHI3 was detected in the culture supernatant, and was purified to homogeneity by three-step chromatography. Amino-acid sequence analysis showed that processing by the signal peptidase was carried out exactly at the expected site. Measurements of circular dichroism spectra and inhibitory activity indicated that OMCHI3 was produced in the properly folded form. Furthermore, site-specific replacement of the Ala residue at the P1 site with Met or Lys resulted in acquisition of inhibitory activity toward chymotrypsin or trypsin, respectively, indicating that the P1 site is the predominant determinant for inhibitory specificity.

Vitamin carrier proteins during embryonic development in birds and mammals.

Egg maturation in oviparous vertebrates involves the hepatic synthesis, secretion, and deposition in the developing oocyte of several maternal proteins with specific nutrient carrier function. Thus, in the chicken, adequate yolk deposition of riboflavin, thiamin, etc. is obligatorily mediated by carrier proteins specific to each vitamin. Like vitellogenin, these are oestrogen-inducible specific gene products. Despite differences in patterns of embryonic development in mammals vis-à-vis oviparous species, immunologically and biochemically similar maternal vitamin carriers participate in the transplacental transport and fetal accumulation of these vitamins during gestation in the rat. The rodent riboflavin and thiamin carrier proteins are also oestrogen-induced maternal proteins of hepatic origin. Their functional importance in fetal development was established by in vivo passive immunoneutralization of the endogenous proteins, which precipitated fetal wastage leading to pregnancy termination, due to curtailment of the vitamin supply to the fetuses. Similarly, active immunization of female rats with the vitamin carrier proteins led to early fetal resorption without interference with maternal health, cyclicity and fecundity. The discovery of similar gestation-specific carrier proteins in higher mammals and humans suggests that carrier-mediated vitamin delivery mechanisms ensuring embryonic growth have been conserved during evolution.

Penguin egg-white and polar fish blood-serum proteins.

The development of, and findings in, a long-term research program on penguin proteins and polar fish blood proteins are described. Two of the egg-white proteins from the Adelie penguin (Pygoscelis adeliae) have unique properties: a glycoprotein named penalbumin that is a major constituent with some characteristics similar to ovalbumin, and an ovomucoid with strong inhibitory capacity for subtilisin as well as for bovine trypsin and alpha-chymotrypsin. The antifreeze glycoproteins from Antarctic fish (Trematomus borchgrevinki and Dissostichus mawsoni) and an Arctic fish (Boreogadus saida) appear to function noncolligatively by lowering the freezing temperature without affecting the melting point. Current evidence indicates that the antifreeze glycoprotein functions at the ice-solution interface, either on the ice surface or in a transition layer between the solution and the ice.