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Phillips W. Robbins Boston, MA 02118-2394 |
education
Ph.D. University of Illinois, 1955
Postdoctoral Training: Rockefeller University, 1956-1959
research description
chitin synthesis
In Saccharomyces cerevisiae most chitin is synthesized by Chs3p, which deposits chitin in the lateral cell wall and in the bud-neck region during cell division. We have recently found that addition of glucosamine (GlcN) to the growth medium leads to a three- to fourfold increase in cell wall chitin levels. We compared this result to the increases in cellular chitin levels associated with cell wall stress and with treatment of yeast with mating pheromone. Since all three phenomena lead to increases in precursors of chitin, we hypothesized that chitin synthesis is at least in part directly regulated by the size of this pool. This hypothesis was strengthened by our finding that addition of GlcN to the growth medium causes a rapid increase in chitin synthesis without any pronounced change in the expression of more than 6,000 genes monitored with Affymetrix gene expression chips. In other studies we found that the specific activity of Chs3p is higher in the total membrane fractions from cells grown in GlcN and from mutants with weakened cell walls. Sucrose gradient analysis shows that Chs3p is present in an inactive form in what may be Golgi compartments but as an active enzyme in other intracellular membrane-bound vesicles, as well as in the plasma membrane. We conclude that Chs3p-dependent chitin synthesis in S. cerevisiae is regulated both by the levels of intermediates of the UDP-GlcNAc biosynthetic pathway and by an increase in the activity of the enzyme in the plasma membrane.
N-Linked Glycosylation in Protists (collaboration with John Samuelson)
The vast majority of eukaryotes (fungi, plants, animals, slime mold, and euglena) synthesize Asn-linked glycans (Alg) by means of a lipid-linked precursor dolichol-PP-GlcNAc2Man9Glc3. Knowledge of this pathway is important because defects in the glycosyltransferases (Alg1-Alg12 and others not yet identified), which make dolichol-PP-glycans, lead to numerous congenital disorders of glycosylation. We used bioinformatic and experimental methods to characterize Alg glycosyltransferases and dolichol-PP-glycans of diverse protists, including many human pathogens, with the following major conclusions. First, it is demonstrated that common ancestry is a useful method of predicting the Alg glycosyltransferase inventory of each eukaryote. Second, in the vast majority of cases, this inventory accurately predicts the dolichol-PP-glycans observed. Third, Alg glycosyltransferases are missing in sets from each organism (e.g., all of the glycosyltransferases that add glucose and mannose are absent from Giardia and Plasmodium). Fourth, dolichol-PP-GlcNAc2Man5 (present in Entamoeba and Trichomonas) and dolichol-PP- and N-linked GlcNAc2 (present in Giardia) have not been identified previously in wild-type organisms. Finally, the present diversity of protist and fungal dolichol-PP-linked glycans appears to result from secondary loss of glycosyltransferases from a common ancestor that contained the complete set of Alg glycosyltransferases.
representative publications
Magnelli P, Cipollo JF, Ratner DM, Cui J, Kelleher D, Gilmore R, Costello CE, Robbins PW, and Samuelson JC. Unique ASN-linked oligosaccharides of the human pathogen entamoeba histolytica. J Biol Chem. 2008 Apr 16. [Epub ahead of print]
Banerjee S, Cui J, Robbins PW, Samuelson J. Use of Giardia, which appears to have a single nucleotide-sugar transporter, to identify the UDP-Glc transporter of Entamoeba. Mol. Biochem. Parasitol. 2008 159:44-53.
Banerjee S, Magnelli P, Vishwanath P, Robbins PW, Samuelson J. Evolution of quality control of protein-folding in the ER lumen. Proc. Natl. Acad. Sci. USA 2007,104:11676-81.
Prenylation of Saccharomyces Cerevisiae Chs4p Affects Chitin Synthase III Activity and Chitin Chain Length. Grabinska KA, Magnelli P, Robbins PW. Eukaryot Cell 6 (2): 328-36 (2007)
Das S, VanDellen K, Bulik D, Magnelli P, Cui J, Head J, Robbins PW, Samuelson J. The cyst wall of Entamoeba invadens contains chitosan (deacetylated chitin). Mol. Biochem. Parasotol. 2006, 148:86-92.
Van Dellen KL, Chatterjee A, Ratner D, Magnelli PE, Cipollo J, Robbins PW, Samuelson J. Chitin-binding lectins of Entamoeba invadens cyst walls have unique post-translational modifications. Eukaryotic Cell 2006; 5:836-48.
Van Dellen KL, Bulik DA, Specht CA, Robbins PW, Samuelson JC. Heterologous expression of an Entamoeba histolytica chitin synthase in Saccharomyces cerevisiae. Eukaryot Cell 2006; 5:203-206.
Robbins PW, Samuelson J. Asparagine linked glycosylation in Giardia. Glycobiology 2005; 15:15G-16G.
Magnelli PE, Cipollo JF, Robbins PW. A glucanase-driven fractionation allows redefinition of Schizosaccharomyces pombe cell wall composition and structure: assignment of diglucan. Anal. Biochem. 2005; 336:202-212.
Samuelson J, Banerjee S, Magnelli P, Cui J, Kelleher DJ, Gilmore R, Robbins PW. The diversity of dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases. Proc Natl Acad Sci USA 2005; 102:1548-1553.