We have reported that a substantial fraction of N-Linked glycans released by Peptide:N glycosidase F (PNGaseF) from bovine lung glycoproteins carry negative charges due to carboxylic acids that are not sialic acids (Norgard-Sumnicht et. al, J. Biol. Chem. 270:27634, 1995). We also used hydrazinolysis to release a "library" of oligosaccharides from the same tissue, and found a much greater yield of N-linked chains than with PNGaseF. This implies PNGaseF-resistance, which is reportedly due to core alpha-1,3 fucosylation in plants and invertebrates.

A portion of the released glycans were radiolabeled at the reducing terminus with [³H]NaBH. After enzymatic or chemical desialylation, the majority of these glycans retained a negative charge that was only partly attributable to sulfate or phosphate esters. Similar to their PNGaseF-released counterparts, many of these desialylated glycans carry carboxylic acid groups, as demonstrated by neutralization with methyl iodide or diazomethane treatment. While lectin binding and compositional analyses indicated that most were complex-type N-linked chains, glucuronic acid residues were not detected, and mixed exoglycosidase digestions failed to release the negative charge, even when beta-glucuronidase or alpha-iduronidasewe re included. To explore these unusual carboxylate groups, we prepared a defined fraction of reduced glycans that had shifted to a single negative charge after desialylation. We then introduced ³H into the molecules using an optimized methyl esterification-[³H]NaBH reduction procedure which converts the carboxylate groups into ³H alcohols. Methyl esterification-dependent introduction of ³H label and loss of negative charge confirmed the validity of the approach. Strong acid hydrolysis of these neutral radiolabeled glycans yielded fragments which eluted in the position of di- or tri-saccharides, and were resistant to further hydrolysis. Both re-N-acetylation and treatment with NHS-biotin indicate that these hydrolysis fragments contain free amino groups. Further studies are underway to determine their native structure.

To further fractionate the bovine N-glycans as well as explore their biology, they were tagged at their reducing terminus with a biotinylated fluorescent tag (Biotinylated Diaminopyridine, BAP). These tagged glycans were fractionated by several orthogonal HPLC techniques. Exoglycosidase digestions showed terminal alpha-Gal residues and unknown modifications (likely the carboxylate residues) which prevent trimming. When the biotinylated exoglycosidase-resistant glycans were complexed to streptavidin and used to immunize mice, a strong antibody response to bovine lung proteins was seen. This allowed us to generate and select IgG mouse mAbs whose binding to bovine lung proteins was blocked by lung glycopeptides in a manner dependent upon the carboxylate residues. Western blotting studies confirmed the presence of these residues on certain glycoproteins from the lung, as well as from several human tissues (brain, heart, pancreas, ovary, etc.). Thus, a large fraction of bovine lung oligosaccharides contain unusual modifications including unexplained carboxylate groups and possibly core alpha-1,3 fucose residues. Antibody studies indicate that they may be widespread in mammalian tissues.

PUBLICATIONS (resulting from this training, and some recent ones)

Norgard-Sumnicht KE, Roux L, Toomre DK, Manzi A, Freeze HH, Varki A. (1995) Unusual anionic N-linked oligosaccharides from bovine lung. J Biol Chem. 270:27634-45.

Toomre D, Keller P, White J, Olivo JC, Simons K. (1999) Dual-color visualization of trans-Golgi network to plasma membrane traffic along microtubules in living cells. J Cell Sci. 112:21-33.

Toomre D, Steyer JA, Keller P, Almers W, Simons K. (2000) Fusion of constitutive membrane traffic with the cell surface observed by evanescent wave microscopy. J Cell Biol. 149:33-40. Simons K, Toomre D. (2001) Lipid rafts and signal transduction. Nat Rev Mol Cell Biol. 1:31-9.

Srikrishna G, Toomre DK, Manzi A, Panneerselvam K, Freeze HH, Varki A, Varki NM. (2001) A novel anionic modification of N-glycans on mammalian endothelial cells is recognized by activated neutrophils and modulates acute inflammatory responses. J Immunol. 166:624-32.

Keller P, Toomre D, Diaz E, White J, Simons K. (2001) Multicolour imaging of post-Golgi sorting and trafficking in live cells. Nat Cell Biol. 3:140-9.

Toomre D, Manstein DJ. (2001) Lighting up the cell surface with evanescent wave microscopy. Trends Cell Biol. 11:298-303.

Rustom A, Bajohrs M, Kaether C, Keller P, Toomre D, Corbeil D, Gerdes HH. (2002) Selective delivery of secretory cargo in Golgi-derived carriers of nonepithelial cells. Traffic 3:279-88.

Pelletier L, Stern CA, Pypaert M, Sheff D, Ngo HM, Roper N, He CY, Hu K, Toomre D, Coppens I, Roos DS, Joiner KA, Warren G. (2002) Golgi biogenesis in Toxoplasma gondii. Nature 418:548-52.

Chow A, Toomre D, Garrett W, Mellman I. (2002) Dendritic cell maturation triggers retrograde MHC class II transport from lysosomes to the plasma membrane. Nature 418:988-94.

Krylyshkina O, Anderson KI, Kaverina I, Upmann I, Manstein DJ, Small JV, Toomre DK. (2003) Nanometer targeting of microtubules to focal adhesions. J Cell Biol. 161:853-9.

He CY, Ho HH, Malsam J, Chalouni C, West CM, Ullu E, Toomre D, Warren G. Golgi duplication in Trypanosoma brucei. (2004) J Cell Biol. 165:313-21.

Cestra G, Toomre D, Chang S, De Camilli P. (2005) The Abl/Arg substrate ArgBP2/nArgBP2 coordinates the function of multiple regulatory mechanisms converging on the actin cytoskeleton. Proc Natl Acad Sci USA 102:1731-6.

Gong LW, Di Paolo G, Diaz E, Cestra G, Diaz ME, Lindau M, De Camilli P, Toomre D. (2005) Phosphatidylinositol phosphate kinase type I gamma regulates dynamics of large dense-core vesicle fusion. Proc Natl Acad Sci USA 102:5204-9.

Toomre D. (2005) Spying on IgE receptor signaling: simply complex, or not? J Cell Biol. 171:415-7.

Millan J, Hewlett L, Glyn M, Toomre D, Clark P, Ridley AJ. (2006) Lymphocyte transcellular migration occurs through recruitment of endothelial ICAM-1 to caveola- and F-actin-rich domains. Nat Cell Biol. 8:113-23.

Hua W, Sheff D, Toomre D, Mellman I. (2006) Vectorial insertion of apical and basolateral membrane proteins in polarized epithelial cells revealed by quantitative 3D live cell imaging. J Cell Biol. 172:1035-44.

Murata T, Delprato A, Ingmundson A, Toomre DK, Lambright DG, Roy CR. (2006) The Legionella pneumophila effector protein DrrA is a Rab1 guanine nucleotide-exchange factor. Nat Cell Biol. 8:971-7.

Perera RM, Zoncu R, Lucast L, De Camilli P, Toomre D. (2006) Two synaptojanin 1 isoforms are recruited to clathrin-coated pits at different stages. Proc Natl Acad Sci USA 103:19332-7.

Iwakiri Y, Satoh A, Chatterjee S, Toomre DK, Chalouni CM, Fulton D, Groszmann RJ, Shah VH, Sessa WC. (2006) Nitric oxide synthase generates nitric oxide locally to regulate compartmentalized protein S-nitrosylation and protein trafficking. Proc Natl Acad Sci USA 103:19777-82.

Zoncu R, Perera RM, Sebastian R, Nakatsu F, Chen H, Balla T, Ayala G, Toomre D, De Camilli PV. (2007) Loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate. Proc Natl Acad Sci USA 104:3793-8.