Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.5.1.52 (PNGase F)
 1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We studied the structures of four N-linked oligosaccharide chains of the recombinant human antithrombin (rAT) expressed in the yeast Pichia pastoris. rAT was fully glycosylated at Asn 96 and Asn 155, whereas the glycosylation on Asn 135 and Asn 192 was partial. The glycosylation level on Asn 135 was only 12% and this reduction is assumed to be one of the reasons for a higher heparin-binding affinity of rAT than plasma-derived human antithrombin (pAT). In order to determine the sizes and electrostatic charges of the N-linked oligosaccharides, rAT was treated with PNGase F, and the reduced ends were labelled by pyridylamination followed by analysis using anion exchange and amide adsorption columns. The N-linked oligosaccharides were 78% neutral and 22% phosphomannosylated. The neutral oligosaccharides were thought to be Man(9-12)GlcNAc(2) as their major components. The phosphomannosylated oligosaccharides were then subjected to mild acid hydrolysis and/or digestion with alkaline phosphatase, and their charge shifts were analysed by the affinity to an anion exchange column. Among phosphomannosylated oligosaccharides, monophosphate diester type was predominant, whereas negatively charged diphosphate diester and monophosphate monoester types were minor components. The mannose residues at the non-reducing end(s) of Man(9-12)GlcNAc(2) were phosphomannosylated or phosphorylated and these are the major components. Because rAT is less negatively charged than pAT, which has disialyl biantennary N-glycans, it might be less repulsive to pentasaccharide-bearing anticoagulantly active heparan sulphate proteoglycan molecules exposed on the surface of the damaged vascular vessels.
 ...
PMID:Characterization of N-linked oligosaccharides attached to recombinant human antithrombin expressed in the yeast Pichia pastoris. 1227 56

Human plasma-derived antithrombin was characterized in both the native and de-N-glycosylated forms (without separation of isoforms) by means of electrospray ionization ion trap mass spectrometry (ESI-ITMS). In order to determine the limits of the instrument set-up, the molecular mass precision and accuracy of the ESI-ITMS analysis was evaluated with the standard protein enolase and some instrumental data acquisition parameters were optimized. Mass precision was determined as a function of the number of averaged mass spectra (= scans) and data acquisition time. For this study, 20 and 50 scans were averaged and the data acquisition time was chosen to be between 0.5 and 5 min. It turned out that data acquisition times longer than approximately 2 min show no significant differences of the standard deviation of the determined molecular mass. Furthermore, the ion trap scan rate was varied at constant acquisition time of 2 min and the number of averaged scans was set to 20. At the scan rate of 13,000 u s(-1) a mass precision of +/-1.8 Da and a mass accuracy of +0.026% were determined. On reducing the scan rate to 5500 u s(-1), better agreement with the theoretical molecular mass was obtained, showing a mass accuracy of +0.012% but with a decrease in the mass precision to +/-3.0 Da. Using the optimized scan rate of 13,000 u s(-1) and a data acquisition time of 2 min, the exact molecular mass was determined of the three forms of antithrombin, namely the alpha-form, the beta-form and the natural mixture (present in human plasma) containing both forms. The protonated molecular masses were found to be 57,854 and 55,664 Da for the affinity chromatography-isolated alpha-and beta-form, respectively. The mass difference of 2190 Da is attributed to the known difference in carbohydrate content at one specific site. The protonated molecular mass of the dominating species of the natural mixture in human plasma was shown to be 57,850 Da, corresponding to the alpha-form, the major component in native plasma. In this mixture the beta-form was also detected, exhibiting a protonated molecular mass of 55,655 Da, but showing a much lower abundance, as expected. To obtain a complete release of the N-glycan residues by means of PNGase F, a denaturation, reduction and alkylation step of the glycoproteins was performed before the enzymatic reaction. After enzymatic removal of all N-glycans, the protonated molecular masses obtained were 49,399, 49,380 and 49,391 Da for the alpha-form, the beta-form and the unseparated natural mixture, respectively. These values are in good agreement (+0.026% for the alpha-form, -0.012% for the beta-form and +0.010% for the unseparated mixture) with the calculated molecular mass based on the SwissProt data. The determined molecular masses after reduction/alkylation and de-N-glycosylation of the alpha-and beta-forms are almost equal, indicating that no major differences exist between the three preparations on the amino acid level.
 ...
PMID:Exact molecular mass determination of various forms of native and de-N-glycosylated human plasma-derived antithrombin by means of electrospray ionization ion trap mass spectrometry. 1557 42

BJ-48, a serine protease from the venom of Bothrops jararacussu, was purified to homogeneity using affinity chromatography on p-aminobenzamidine-agarose followed by HPLC gel filtration. BJ-48 presented 52kDa by SDS-PAGE analysis and 48,036Da by electron spray mass spectrometry. The enzyme was shown to be highly glycosylated with 42% of N-linked carbohydrates composed of Fuc(1):GalN(4):GlcN(5):Gal(1):Man(2) and a high content of sialic acid residues (8-12%). BJ-48 had optimal esterase activity at pH 7.5 and displayed maximum catalytic rate at 50 degrees C. Its hydrolytic activity was strongly inhibited by aprotinin and dithiothreitol while N-tosyl-l-phenylalanine chloromethyl ketone, 6-aminocaproic acid, E-64 and soybean trypsin inhibitor (SBTI) were ineffective. The kinetics of BJ-48 with chromogenic substrates revealed an unprecedented selectivity (10(4)-fold) for Arg over Lys in P1. BJ-48 proved to be a thrombin-like enzyme (TLE) with a specific fibrinogen-clotting activity of 73.4NIH units/mg. The TLE rapidly digested human fibrinogen Bbeta chain, but the Aalpha chain was cleaved specifically to release fibrinopeptide A with k(cat)/K(m)=2.1 microM(-1)s(-1). The TLE showed no activity toward other thrombin substrates like protein C, protease-activated receptor-1 or inhibitors such as hirudin and antithrombin. A non-denaturing procedure using PNGase F and neuraminidase followed by hydrophobic interaction chromatography was employed to obtain active BJ-48 forms with variable carbohydrate content. Compared to the native enzyme, total or partially deglycosylated BJ-48 forms presented up to 2-fold reduction in their specific activities upon heating at 55/65 degrees C or treatment with SBTI. These results point out a role for BJ-48 glycosylation in thermostability and controlling the access of some canonical protein inhibitors to the active site.
 ...
PMID:BJ-48, a novel thrombin-like enzyme from the Bothrops jararacussu venom with high selectivity for Arg over Lys in P1: Role of N-glycosylation in thermostability and active site accessibility. 1743 97