Although the importance of glycosylation has been thoroughly recognized in association with a number of biological processes, efficient assessments of glycans have been hampered by both the limited size of specimens and lengthy sample preparations, particularly in clinical settings. Here we report a simple preparative method for N-glycan analyses. It involves only short one-step chloroform-methanol extraction in presence or absence of water prior to PNGase F deglycosylation. The procedure was successfully applied to the investigation of N-glycans obtained from small numbers of in vitro cultured cancer cells (≤1 × 10(5)) and to tumor tissues, including patient biopsies of small size. MALDI-MS analysis confirmed the efficient release of all N-glycan types including complex forms with poly-N-acetyllactosamine chains. In addition, nonaqueous extraction of specimens from several established cancer cell lines, as well as patient tumor tissues, yielded high-mannose glycans with one GlcNAc moiety (Man3-9GlcNAc), strongly suggesting preservation of enzymatic activity analogous to Endo H enzyme. In summary, the method is both a step toward the practical use of glycan profiling and a way to detect Endo H-like activity in cancer specimens.

• MeSH
• glykopeptidasa metabolismus   MeSH
• glykosylace MeSH
• lidé MeSH
• mannosa MeSH
• mannosyl-glykoprotein endo-beta-N-acetylglukosaminidasa metabolismus   MeSH
• nádorové buňky kultivované MeSH
• nádory chemie   patologie   MeSH
• polysacharidy analýza   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• velikost vzorku MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In this paper, we report on a novel oriented peptide-N-glycosidase F (PNGase F) immobilization approach onto methacrylate based monolithic support for rapid, reproducible and efficient release of the N-linked carbohydrate moieties from glycoproteins. The glutathione-S-transferase-fusion PNGase F (PNGase F-GST) was expressed in Escherichia coli using regular vector technology. The monolithic pore surface was functionalized with glutathione via a succinimidyl-6-(iodoacetyl-amino)-hexanoate linker and the specific affinity of GST toward glutathione was utilized for the oriented coupling. This novel immobilization procedure was compared with reductive amination technique commonly used for non-oriented enzyme immobilization via primary amine functionalities. Both coupling approaches were compared using enzymatic treatment of several glycoproteins, such as ribonuclease B, fetuin and immunoglobulin G followed by MALDI/MS and CE-LIF analysis of the released glycans. Orientedly immobilized PNGase F via GST-glutathione coupling showed significantly higher activity, remained stable for several months, and allowed rapid release of various types of glycans (high-mannose, core fucosylated, sialylated, etc.) from glycoproteins. Complete protein deglycosylation was obtained as fast as in several seconds when using flow-through immobilized microreactors.

• MeSH
• elektroforéza kapilární metody   MeSH
• enzymy imobilizované chemie   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• glykosylace MeSH
• imunoglobulin G chemie   MeSH
• lidé MeSH
• mannosyl-glykoprotein endo-beta-N-acetylglukosaminidasa chemie   metabolismus   MeSH
• polysacharidy analýza   chemie   MeSH
• poréznost MeSH
• skot MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Fungal β-N-acetylhexosaminidases are inducible extracellular enzymes with many biotechnological applications. The enzyme from Penicillium oxalicum has unique enzymatic properties despite its close evolutionary relationship with other fungal hexosaminidases. It has high GalNAcase activity, tolerates substrates with the modified N-acyl group better and has some other unusual catalytic properties. In order to understand these features, we performed isolation, biochemical and enzymological characterization, molecular cloning and molecular modelling. The native enzyme is composed of two catalytic units (65 kDa each) and two propeptides (15 kDa each), yielding a molecular weight of 160 kDa. Enzyme deglycosylated by endoglycosidase H had comparable activity, but reduced stability. We have cloned and sequenced the gene coding for the entire hexosaminidase from P. oxalicum. Sufficient sequence identity of this hexosaminidase with the structurally solved enzymes from bacteria and humans with complete conservation of all catalytic residues allowed us to construct a molecular model of the enzyme. Results from molecular dynamics simulations and substrate docking supported the experimental kinetic and substrate specificity data and provided a molecular explanation for why the hexosaminidase from P. oxalicum is unique among the family of fungal hexosaminidases.

• MeSH
• beta-N-acetylhexosaminidasy chemie   genetika   izolace a purifikace   metabolismus   MeSH
• fungální proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• glykosylace MeSH
• katalytická doména MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• konzervovaná sekvence MeSH
• mannosyl-glykoprotein endo-beta-N-acetylglukosaminidasa metabolismus   MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• Penicillium enzymologie   genetika   MeSH
• prekurzory enzymů chemie   genetika   izolace a purifikace   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• sekvenční seřazení MeSH
• simulace molekulární dynamiky MeSH
• stabilita enzymů MeSH
• substrátová specifita MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH