N-glycosylation of proteins is now routinely characterized and monitored because of its significance to the detection of disease states and the manufacturing of biopharmaceuticals. At the same time, hydrophilic interaction chromatography (HILIC) has emerged as a powerful technology for N-glycan profiling. Sample preparation techniques for N-glycan HILIC analyses have however tended to be laborious or require compromises in sensitivity. To address these shortcomings, we have developed an N-glycan labeling reagent that provides enhanced fluorescence response and MS sensitivity for glycan detection and have also simplified the process of preparing a sample for analysis. The developed labeling reagent rapidly reacts with glycosylamines upon their release from glycoproteins. Within a 5 min reaction, enzymatically released N-glycans are labeled with this reagent comprised of an NHS-carbamate reactive group, a quinoline fluorophore, and a tertiary amine for enhancing ESI+ MS ionization. To further expedite the released N-glycan sample preparation, rapid tagging has been integrated with a fast PNGase F deglycosylation procedure that achieves complete deglycosylation of a diverse set of glycoproteins in approximately 10 min. Moreover, a technique for HILIC-SPE of the labeled glycans has been developed to provide quantitative recovery and facilitate immediate HILIC analysis of the prepared samples. The described approach makes it possible to quickly prepare N-glycan samples and to incorporate the use of a fluorescence and MS sensitivity enhancing labeling reagent. In demonstration of these new capabilities, we have combined the developed sample preparation techniques with UHPLC HILIC chromatography and high sensitivity mass spectrometry to thoroughly detail the N-glycan profile of a monoclonal antibody.

Bacteroides-Escherichia coli shuttle vectors containing a nonmobilizable pBR322 derivative and either pBFTM10 (pDP1, pCG30) or pB8-51 (pEG920) were mobilized by IncP plasmid R751 or pRK231 (an ampicillin-sensitive derivative of RK2) between E. coli strains and from E. coli to Bacteroides recipients. IncIaR64 drd-11 transferred these vectors 1,000 times less efficiently than did the IncP plasmids. pDPl, pCG30, and pEG920 could be mobilized from B. uniformis donors to (8,9,19,20

In an effort to identify novel endo-α-N-acetylgalact- osaminidases (endo-α-GalNAcases), four potential genes were cloned. Three of the expressed proteins EngEF from Enterococcus faecalis, EngPA from Propionibacterium acnes, and EngCP from Clostridium perfringens were purified and characterized. Their substrate specificity was investigated and compared to the commercially available endo-α-GalNAcases from Streptococcus pneumoniae (EngSP) and Alcaligenes sp. (EngAL). All enzymes were incubated with various synthetic substrates, and natural glycoproteins and the released sugars were detected by colorimetric assay and thin layer chromatography analysis. The Core 1 disaccharide Galβ1,3GalNAcα1pNP was the most rapidly hydrolyzed substrate by all enzymes tested. EngEF exhibited the highest kcat for this substrate. EngEF and EngPA were also able to fully hydrolyze the Core 3 disaccharide GlcNAcβ1,3GalNAcα1pNP. This is the first report of endo-α-GalNAcases EngEF and EngPA acting on Core 3 in addition to Core 1 O-glycans. Interestingly, there were no significant differences in transglycosylation activities when Galβ1,3GalNAcα1pNP or GlcNAcβ1,3GalNAcα1pNP was incubated with various 1-alkanols in the presence of the endo-α-GalNAcases tested in this work.

No abstract

Bacteroides are Gram-negative, obligate anaerobes that are present in high concentrations within the intestinal tracts of humans and animals. Bacteroides are also important opportunistic pathogens of humans and animals. Methods for genetic manipulation of these important organisms have only recently begun to emerge. Shuttle vectors which can be transferred by conjugation between Escherichia coli to Bacteroides are now available. A method for transforming some strains of Bacteroides has been developed. Two Bacteroides transposons, Tn4351 and Tn4400, have been found and one of them, Tn4351, has been used for transposon mutagenesis of Bacteroides. Several different Bacteroides genes have now been cloned, including a gene that codes for resistance to clindamycin, genes that code for polysaccharidases (chondroitin lyase and pullulanase), and a gene that codes for a fimbrial subunit. These cloned genes have been used to study the organization and regulation of Bacteroides genes.