Release of bifidogenic N-glycans from native bovine colostrum proteins by an endo-ß-N-acetylglucosaminidase.

Milk glycoproteins play various biological roles including antibacterial, antiviral activities, modulating immune responses in living organisms. Released N-glycans from milk glycoproteins act as growth substrates for infant-associated bifidobacteria, which are key members of the breastfed infant's gut. To date, the mechanisms, and contributions of glycans to the biological activities of glycoproteins remain to be elucidated. Only by testing both the released glycans and the deglycosylated protein in their native (i.e., non-denatured) form, can the individual contribution to the biological activity of glycoproteins be elucidated. However, for conventional enzymatic and chemical deglycosylation strategies to work efficiently, glycoprotein denaturation is required, which alters the protein native shape, hindering further investigations of its biological roles. An endo-ß-N-acetylglucosaminidase (EndoBI-1) from Bifidobacterium longum subsp. infantis ATCC 15697 (B. infantis) was characterized as having the ability to release N-glycans from bovine milk glycoproteins efficiently, without the denaturation. In this study, the activity of EndoBI-1 was compared to a commercial enzyme to release N-glycans, the peptide-N-glycosidase F (PNGase F), using dairy glycoproteins as the substrate. The kinetic evaluation showed that EndoBI-1 displayed higher activity on native glycoproteins than PNGase F, with 0.036 mg/mL×min and 0.012 mg/mL×min glycan release, respectively. EndoBI-1 released a broader array of glycan structures compared to PNGase F from native glycoproteins. Thirty-two and fifteen distinct compositions were released from the native glycoproteins by EndoBI-1 and PNGase F, respectively, as characterized by advanced mass spectrometry. EndoBI-1 can be considered a promising enzyme for the release of N-glycans and their protein backbone in the native form, which will enable effective glycan release and will facilitate subsequent investigations to reveal their contribution to glycoproteins' biological roles.

Water renal management is altered more frequently than albuminuria in children in the G1 stage of the 2012 KDIGO Guideline.

BACKGROUND: The G1 stage of chronic kidney disease (CKD) is defined in the 2012 KDIGO Guideline as kidney damage characterized by structural or functional kidney abnormalities without deterioration of glomerular filtration rate. Albuminuria and electrolyte abnormalities due to tubular disorders are considered functional markers of kidney damage. Changes in renal water handling are not explicitly cited in these guidelines. A large sample of children with abnormal dimercaptosuccinic acid (DMSA) scan located in the G1 stage was used in this study. METHODS: Ambispective, cross-sectional study to evaluate the clinical histories of 116 pediatric patients. 100 patients were included in the first group (G1 stage) and 16 patients in the G2-G5 stages according to the classification of CKD Guideline KDIGO. All the patients had a renal pathologic DMSA scan. GFR, maximum urine osmolality and albumin/creatinine and NAG/creatinine ratios were determined. RESULTS: The patients with normal GFR, in relation to those with reduced GFR, had significantly higher values of maximum urine osmolality and significantly reduced values of urine volume and albumin/creatinine and NAG/creatinine ratios. The most frequently observed alterations in children in the KDIGO G1 stage were those involving the water renal management such as urinary concentrating ability defect (29%) and increased urinary volume (20%). The frequency of children with increased urinary elimination of albumin (12%) and NAG (3%) was more lower. All children in KDIGO G2-G5 stages had alterations in water renal management. CONCLUSIONS: The parameters related with the water renal management are affected more frequently than albumin urinary excretion in children who have loss of parenchyma and normal GFR.

A novel fluorescence-based assay for the transglycosylation activity of endo-beta-N-acetylglucosaminidases.

A fluorescence-based assay for the transglycosylation activity of endo-beta-N-acetylglucosaminidases (ENGases) was developed. The assay was based on the findings that a coupled chitinase can specifically capture and hydrolyze the fluorogenic intermediate that is formed by the ENGase-catalyzed transglycosylation to release a fluorophore, but does not hydrolyze the donor asparagine-linked N-glycan and the acceptor 4-methylumbelliferyl N-acetylglucosaminide. The assay method was verified by detecting the transglycosylation activities of the known ENGases. Its application for assessing the effects of organic solvents on transglycosylation activity was demonstrated. The novel coupled assay provides a highly sensitive, easy, and quantitative method for screening endo-beta-N-acetylglucosaminidases with transglycosylation activities useful for glycoconjugate synthesis.

Subcellular localization of endo-beta-N-acetylglucosaminidase and high-mannose type free N-glycans in plant cell.

Subcellular distribution of plant endo-beta-N-acetylglucosaminidase (endo-beta-GlcNAc-ase) and high-mannose type free N-glycans produced by the endoglycosidase has been analyzed using cotyledons of pumpkin seedlings as the model plant cells. Each organelle in the cotyledons was fractionated by ultracentrifugation with the sucrose density gradient system and the endo-beta-GlcNAc-ase activity in each fraction was assayed with fluorescence labeled N-glycans as substrates. The endoglycosidase activity was exclusively recovered in the soluble fraction (cytosol fraction) but not in other specific organellar fractions, suggesting that the endoglycosidase would reside predominantly in the cytosol. The quantitative analysis of high-mannose type free N-glycans occurring in each fraction showed that more than 70% of the free N-glycans was recovered from the soluble fraction, suggesting the endoglycosidase would work in the cytosol and the resulting free N-glycans would accumulate in the same fraction. The pumpkin endo-beta-GlcNAc-ase (endo-CM) partially purified from the cotyledons showed optimum activity around pH 6.5, supporting this enzyme would reside in the cytosol. Furthermore, the detailed analysis of substrate specificity of endo-CM using various high-mannose type N-glycans showed that the pumpkin enzyme, as well as other plant endo-beta-N-acetylglucosaminidases, were highly active toward the high-mannose type glycans bearing the Man(alpha1)-2Man(alpha1)-3Man(beta1)-structural unit.

Assay of glycoamidases and endo-beta-N-acetylglucosaminidases by lectin capture and dissociation-enhanced lanthanide fluorescence immunoassay.

We have developed an assay system for endo-beta-N-acetylglucosaminidase and glycoamidase (PNGase), using Eu(3+)-labeled Man(9)GlcNAc(2) glycopeptides as substrates in combination with lectin capture. Two glycopeptides of different peptide lengths, derived from soybean agglutinin, were labeled with Eu(3+) via a diethylenetriaminepentaacetate (DTPA) chelating linker and served as substrates for two types of enzymes: one with (Man(9)GlcNAc(2))Asn for endo-beta-N-acetylglucosaminidase and the other with Ala-Ser-Phe-(Man(9)GlcNAc(2))Asn-Phe-Thr for glycoamidase activities. Following enzymatic hydrolysis, concanavalin A, immobilized or soluble, was added to the mixture to bind unreacted substrate and unlabeled hydrolysis product. The labeled peptide product could then be separated from the lectin-bound complexes by filtration for quantification by dissociation-enhanced lanthanide fluorescence immunoassay. Activities as low as 2 fmol min(-1) could be rapidly quantified for both types of enzymes, and enzymological parameters could be determined within minutes. Applicability of the assay was tested for identification of a glycoamidase activity peak in the fractionation of sweet almond emulsin, a classic example. This assay offers sensitivity, ease of use, and high throughput. In addition, it is versatile and should be applicable to other glycobiology enzyme systems.

A fluorescence high-performance liquid chromatography assay for enzymes acting on the di-N-acetylchitobiosyl part of asparagine-linked glycans.

The glycoasparagine, Man7GlcNAc2Asn ('Man7') was labelled with resorufin and used as a specific substrate for the detection and quantification of endo-beta-N-acetyl glucosaminidases (Endos) acting on the di-N-acetylchitobiosyl part of asparagine-linked glycans. Peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidases (PNGases) cannot transform this substrate but they can be detected by the procedure described earlier using the resorufin-labelled N-glycopeptide [Glycoconjugate J., 9 (1992) 162-167]. These two substrates can be used in a simple, reproducible and very sensitive fluorescence HPLC assay in order to monitor Endo and PNGase activities during isolation and purification processes, or studies of the evolution of such activities during cultivation of the producing cells.