Phillips Robbins

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.

Publications

Chatterjee, A., Banerjee, S., Steffen, M., O’Connor, R. M., Ward, H. D., Robbins, P. W., Samuelson, J. Evidence for mucin-like glycoproteins that tether sporozoites of Cryptosporidium parvum to the inner surface of the oocyst wall. Eukaryot Cell. 2010 Jan; 9(1): 84–96.

Bushkin, G. G., Ratner, D. M., Cui, J., Banerjee, S., Duraisingh, M. T., Jennings, C. V., Dvorin, J. D., Gubbels, M. J., Robertson, S. D., Steffen, M., O’Keefe, B. R., Robbins, P. W., Samuelson, J. Suggestive Evidence for Darwinian Selection against Asparagine-Linked Glycans of Plasmodium falciparum and Toxoplasma gondii. Eukaryot Cell. 2010 Feb; 9(2): 228–241.

Cui, J., Smith, T., Robbins, P. W., Samuelson, J. Darwinian selection for sites of Asn-linked glycosylation in phylogenetically disparate eukaryotes and viruses. Proc Natl Acad Sci U S A. 2009 Aug 11; 106(32): 13421–6.

Chatterjee, A., Ghosh, S. K., Jang, K., Bullitt, E., Moore, L., Robbins, P. W., Samuelson, J. Evidence for a “wattle and daub” model of the cyst wall of entamoeba. PLoS Pathog. 2009 Jul; 5(7):e1000498.

Banerjee, S., Robbins, P. W., Samuelson, J. Molecular characterization of nucleocytosolic O-GlcNAc transferases of Giardia lamblia and Cryptosporidium parvum. Glycobiology. 2009 Apr; 19(4):331–6.

Ratner, D. M., Cui, J., Steffen, M., Moore, L. L., Robbins, P. W., Samuelson, J. Changes in the N-glycome, glycoproteins with Asn-linked glycans, of Giardia lamblia with differentiation from trophozoites to cysts. Eukaryot Cell. 2008 Nov; 7(11):1930–40.

Grabinska, K. A., Ghosh, S. K., Guan, Z., Cui, J., Raetz, C. R., Robbins, P. W., Samuelson, J. Dolichyl-phosphate-glucose is used to make O-glycans on glycoproteins of Trichomonas vaginalis. Eukaryot Cell. 2008 Aug; 7(8):1344–51.

Magnelli, P., Cipollo, J. F., Ratner, D. M., Cui, J., Kelleher, D., Gilmore, R., Costello, C. E., Robbins, P. W., Samuelson, J. Unique Asn-linked oligosaccharides of the human pathogen Entamoeba histolytica. J Biol Chem. 2008 Jun 27; 283(26):18355–64.

Banerjee, S., Vishwanath, P., Cui, J., Kelleher, D. J., Gilmore, R., Robbins, P. W., Samuelson, J. The evolution of N-glycan-dependent endoplasmic reticulum quality control factors for glycoprotein folding and degradation. Proc Natl Acad Sci U S A. 2007 Jul 10; 104(28):11676–81.

Grabinska, K. A., Magnelli, P., Robbins, P. W. Prenylation of Saccharomyces cerevisiae Chs4p Affects Chitin Synthase III activity and chitin chain length. Eukaryot Cell. 2007 Feb; 6(2):328–36.