The degradation of many proteins involves the sequential ligation of ubiquitin molecules to the substrate to form a multiubiquitin chain linked through Lys-48 of ubiquitin. To test for the existence of alternate forms of multiubiquitin chains, we examined the effects of individually substituting each of six other Lys residues in ubiquitin with Arg. Substitution of Lys-63 resulted in the disappearance of a family of abundant multiubiquitin-protein conjugates. The UbK63R mutants were not generally impaired in ubiquitination, because they grew at a wild-type rate, were fully proficient in the turnover of a variety of short-lived proteins, and exhibited normal levels of many ubiquitin-protein conjugates. The UbK63R mutation also conferred sensitivity to the DNA-damaging agents methyl methanesulfonate and UV as well as a deficiency in DNA damage-induced mutagenesis. Induced mutagenesis is mediated by a repair pathway that requires Rad6 (Ubc2), a ubiquitinconjugating enzyme. Thus, the UbK63R mutant appears to be deficient in the Rad6 pathway of DNA repair. However, the UbK63R mutation behaves as a partial suppressor of a rad6 deletion mutation, indicating that an effect of UbK63R on repair can be manifest in the absence of the Rad6 gene product. The UbK63R mutation may therefore define a new role of ubiquitin in DNA repair. The results of this study suggest that Lys-63 is used as a linkage site in the formation of novel multiubiquitin chain structures that play an important role in DNA repair.

Ubiquitin is ligated to L28, a component of the large ribosomal subunit, to form the most abundant ubiquitin-protein conjugate in S. cerevisiae. The human ortholog of L28 is also ubiquitinated, indicating that this modification is highly conserved in evolution. During S phase of the yeast cell cycle, L28 is strongly ubiquitinated, while reduced levels of L28 ubiquitination are observed in G1 cells. L28 ubiquitination is inhibited by a Lys63 to Arg substitution in ubiquitin, indicating that L28 is modified by a variant, Lys63-linked multiubiquitin chain. The K63R mutant of ubiquitin displays defects in ribosomal function in vivo and in vitro, including a dramatic sensitivity to translational inhibitors. L28, like other ribosomal proteins, is metabolically stable. Therefore, these data suggest a regulatory role for multiubiquitin chains that is reversible and does not function to target the acceptor protein for degradation.

Enzymatic removal of blood group ABO antigens to develop universal red blood cells (RBCs) was a pioneering vision originally proposed more than 25 years ago. Although the feasibility of this approach was demonstrated in clinical trials for group B RBCs, a major obstacle in translating this technology to clinical practice has been the lack of efficient glycosidase enzymes. Here we report two bacterial glycosidase gene families that provide enzymes capable of efficient removal of A and B antigens at neutral pH with low consumption of recombinant enzymes. The crystal structure of a member of the alpha-N-acetylgalactosaminidase family reveals an unusual catalytic mechanism involving NAD+. The enzymatic conversion processes we describe hold promise for achieving the goal of producing universal RBCs, which would improve the blood supply while enhancing the safety of clinical transfusions.

The highly pleiotropic stage 0 sporulation locus of Bacilus subtilis, spoOA, has been cloned in bacteriophage X, subcloned in plasmids, and sequenced. The locus was found to code for a protein of 29,691 Da. Analysis of the in vivo transcripts from this region by nuclease S1 protection experiments located the start and stop of transcription of the locus. The transcription start site was preceded by a promoter resembling &37-dependent promoters. Two mutations originally assigned to a second locus, spoOC, in this region because of their weakly pleiotropic phenotypes were cloned and sequenced. To study the regulatory mechanisms that control the initiation of sporulation in bacteria, the approach taken in this laboratory has been to isolate mutants defective in the initiation process. These mutants are the stage 0 mutants and the mutations are designated by the genotypic symbol spoO. Nine spoO genes have been found among several hundred mutations mapped (1, 2). The majority of the stage 0 mutations map in five loci that are unlinked on the Bacillus subtilis chromosome. Stage 0 mutants may be blocked in any one of the processes leading to the initiation of development: the formation of an intracellular metabolic signal, the mechanisms by which this signal is associated with the switch mechanism, or the transmission of a metabolic signal to the transcription machinery. In any case it is clear that all of the stage 0 mutants have gross pleiotropic effects on the synthesis of a wide variety of gene products associated with the sporulation process (3). This result may be a consequence of the inability of SpoO mutants to permit transcription from a wide variety of promoters (4, 5). The most pleiotropic of the stage 0 mutations occur in the spoOA locus. In this report, we describe the cloning and sequencing of the spoOA locus. Nuclease S1 Protection Assay. Nuclease S1 mapping of the spoOA locus and the JH703 deletion was performed by the method of Berk and Sharp (10), with some modification. Several single end-labeled DNA probes complementary to the spoOA mRNA were prepared as follows. The EcoRI or Bgl II site of pJF1642 (this plasmid carries the equivalent fragment to pJF1361, plus several hundred bases upstream, in pUC9) were 32P-labeled at their 5' termini by using [y-32P]ATP (ICN, crude, >7000 Ci/mmol; 1 Ci = 37 GBq) and T4 polynucleotide kinase in a reaction previously described (11). The EcoRI site of pJF1599 was 3' end-labeled by using [a-32P]dATP (Amersham; >800 Ci/mmol) and E. coli DNA polymerase I large fragment. End-labeled fragments were isolated from a polyacrylamide gel after digestion at the HindIII site in the polylinker region of the two plasmids. [The sizes of the probes were 1.6 kilobases (kb) from the 5' endlabeled EcoRI fragment, 1.06 kb from the 5' end-labeled Bgl II fragment, and 0.67 kb from the 3' end-labeled EcoRI fragment from pJF1599.] B. subtilis mRNA (100 ,ug) from W168 or JH703 and approximately 40,000 cpm of end-labeled DNA probe were coprecipitated with ethanol, dried briefly under v...

In search of ␣-galactosidases with improved kinetic properties for removal of the immunodominant ␣1,3-linked galactose residues of blood group B antigens, we recently identified a novel prokaryotic family of ␣-galactosidases (CAZy GH110) with highly restricted substrate specificity and neutral pH optimum (Liu, Q. P., Sulzenbacher, G., Yuan, H., Bennett, E. P., Pietz, G., Saunders, K., Spence, J., Nudelman, E., Levery, S. B., White, T., Neveu, J. M., Lane, W. S., Bourne, Y., Olsson, M. L., Henrissat, B., and Clausen, H. (2007) Nat. Biotechnol. 25, 454 -464). One member of this family from Bacteroides fragilis had exquisite substrate specificity for the branched blood group B structure Gal␣1-3(Fuc␣1-2)Gal, whereas linear oligosaccharides terminated by ␣1,3-linked galactose such as the immunodominant xenotransplantation epitope Gal␣1-3Gal␤1-4GlcNAc did not serve as substrates. Here we demonstrate the existence of two distinct subfamilies of GH110 in B. fragilis and thetaiotaomicron strains. Members of one subfamily have exclusive specificity for the branched blood group B structures, whereas members of a newly identified subfamily represent linkage specific ␣1,3-galactosidases that act equally well on both branched blood group B and linear ␣1,3Gal structures. We determined by one-dimensional 1 H NMR spectroscopy that GH110 enzymes function with an inverting mechanism, which is in striking contrast to all other known ␣-galactosidases that use a retaining mechanism. The novel GH110 subfamily offers enzymes with highly improved performance in enzymatic removal of the immunodominant ␣3Gal xenotransplantation epitope.