Denis Growth Hormone

TnBgl1A from the thermophile Thermotoga neapolitana is a dimeric beta-glucosidase that belongs to glycoside hydrolase family 1 (GH1), with hydrolytic activity through the retaining mechanism, and a broad substrate specificity acting on beta-1,4-, beta-1,3- and beta-1,6-linkages over a range of glyco-oligosaccharides. Three variants of the enzyme (TnBgl1A_E349G, TnBgl1A_E349A and TnBgl1A_E349S), mutated at the catalytic nucleophile, were constructed to evaluate their glycosynthase activity towards oligosaccharide synthesis. Two approaches were used for the synthesis reactions, both of which utilized 4-nitrophenyl beta-D-glucopyranoside (4NPGIc) as an acceptor molecule: the first using an alpha-glucosyl fluoride donor at low temperature (35 degrees C) in a classical glycosynthase reaction, and the second by in situ generation of the glycosyl donor with (4NPGIc), where formate served as the exogenous nucleophile under higher temperature (70 degrees C). Using the first approach, TnBgl1A_E349G and TnBgl1A_E349A synthesized disaccharides with beta-1,3-linkages in good yields (up to 61%) after long incubations (15 h). However, the GH1 glycosynthase Bg13_E383A from a mesophilic Streptomyces sp., used as reference enzyme, generated a higher yield at the same temperature with both a shorter reaction time and a lower enzyme concentration. The second approach yielded disaccharides for all three mutants with predominantly beta-1,3-linkages (up to 45%) but also beta-1,4-linkages (up to 12.5%), after 7 h reaction time. The TnBgl1A glycosynthases were also used for glycosylation of flavonoids, using the two described approaches. Quercetin-3-glycoside was tested as an acceptor molecule and the resultant product was quercetin-3,4'-diglycosides in significantly lower yields, indicating that TnBgl1A preferentially selects 4NPGIc as the acceptor. (C) 2014 Elsevier B.V. All rights reserved.

In pre-mRNA splicing, a conserved AG/G at the 3'-splice site is recognized by U2AF(35). A disease-causing mutation abrogating the G nucleotide at the first position of an exon (E(+1)) causes exon skipping in GH1, FECH and EYA1, but not in LPL or HEXA. Knockdown of U2AF(35) enhanced exon skipping in GH1 and FECH. RNA-EMSA revealed that wild-type FECH requires U2AF(35) but wild-type LPL does not. A series of artificial mutations in the polypyrimidine tracts of GH1, FECH, EYA1, LPL and HEXA disclosed that a stretch of at least 10-15 pyrimidines is required to ensure normal splicing in the presence of a mutation at E(+1). Analysis of nine other disease-causing mutations at E(+1) detected five splicing mutations. Our studies suggest that a mutation at the AG-dependent 3'-splice site that requires U2AF(35) for spliceosome assembly causes exon skipping, whereas one at the AG-independent 3'-splice site that does not require U2AF(35) gives rise to normal splicing. The AG-dependence of the 3'-splice site that we analyzed in disease-causing mutations at E(+1) potentially helps identify yet unrecognized splicing mutations at E(+1).