Interconversion of the CD and EF sites in oncomodulin. Influence on the Eu3+ 7F0-->5D0 excitation spectrum

The appearance of the parvalbumin Eu3+ 7F0-->5D0 spectrum is markedly pH dependent, the result of a hitherto unidentified deprotonation event in the CD ion-binding domain [Trevi?o, C.L., et al. (1991) J. Biol. Chem. 265, 9694-9700]. We are studying this phenomenon in the mammalian placental parvalbumin called oncomodulin. As in other parvalbumins, the liganding residues in the CD and EF sites of oncomodulin differ at the +z and -x coordination positions: serine and aspartate, respectively, in the CD site; aspartate and glycine in the EF site. We have prepared a series of oncomodulin variants in which the +z and/or -x residue(s) from one site have been replaced by the corresponding residue(s) from the other. We herein characterize the resulting proteins by Eu3+ luminescence spectroscopy. Simultaneous replacement of serine-55 by aspartate and aspartate-59 by glycine affords the CD site with a coordination sphere superficially equivalent to that of the EF site. As observed previously for the S55D mutation [Henzl, M. T., et al. (1992) FEBS Lett. 314, 130-134], the Eu3+ 7F0-->5D0 spectrum of the 55/59 variant is pH independent. Interestingly, replacement of aspartate-94 by serine at the +z position of the EF site of 55/59 imparts pH dependent behavior to the EF site. The identical mutation in the wild-type background likewise imparts pH dependence to the EF site, affording a protein in which both sites display broad signals near 578.2 nm at pH 8. Significantly, a variant in which threonine replaces serine-55 retains the pH dependent spectroscopic signature. These results indicate that the presence of a hydroxyl group at the +z position is sufficient to confer pH dependence on the 7F0-->5D0 spectrum of a parvalbumin EF-hand domain. Importantly, the data also suggest that the component peaks of the low-pH doublet are not site-specific signals, as previously believed. Rather, they probably represent differences in coordination environment arising from differential hydration or conformational heterogeneity. In wild-type oncomodulin, the CD site signal dominates the low-pH spectrum. Since this dominance persists even when serine-55 and aspartate-59 are replaced by the corresponding EF site residues, it appears that the context of the CD binding site, as dictated by the global polypeptide fold, exerts a major influence on the metal ion-binding properties of the site.     

Architecture of the yeast cell wall. Beta(1-->6)-glucan interconnects mannoprotein, beta(1-->)3-glucan, and chitin

In a previous study (Kollár, R., Petráková, E., Ashwell, G., Robbins, P. W., and Cabib, E. (1995) J. Biol. Chem. 270, 1170-1178), the linkage region between chitin and beta(1-->3)-glucan was solubilized and isolated in the form of oligosaccharides, after digestion of yeast cell walls with beta(1-->3)-glucanase, reduction with borotritide, and subsequent incubation with chitinase. In addition to the oligosaccharides, the solubilized fraction contained tritium-labeled high molecular weight material. We have now investigated the nature of this material and found that it represents areas in which all four structural components of the cell wall, beta(1-->3)-glucan, beta(1-->6)-glucan, chitin, and mannoprotein are linked together. Mannoprotein, with a protein moiety about 100 kDa in apparent size, is attached to beta(1-->6)-glucan through a remnant of a glycosylphosphatidylinositol anchor containing five alpha-linked mannosyl residues. The beta(1-->6)-glucan has some beta(1-->3)-linked branches, and it is to these branches that the reducing terminus of chitin chains appears to be attached in a beta(1-->4) or beta(1-->2) linkage. Finally, the reducing end of beta(1-->6)-glucan is connected to the nonreducing terminal glucose of beta(1-->3)-glucan through a linkage that remains to be established. A fraction of the isolated material has three of the main components but lacks mannoprotein. From these results and previous findings on the linkage between mannoproteins and beta(1-->6)-glucan, it is concluded that the latter polysaccharide has a central role in the organization of the yeast cell wall. The possible mechanism of synthesis and physiological significance of the cross-links is discussed.