Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.1.52 (PNGase F)
1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The extent of N-glycosylation of yeast external invertase at each of the 14 potential sites was determined by the combination of proteolytic digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS). The average molecular mass of the intact external invertase was determined as 97 kDa by MALDI/TOF-MS. The intact protein was digested with trypsin, Lys-C and Asp-N, followed by high-performance liquid chromatographic separation. The proteolytic digests were analyzed by MALDI/MS screening for the glycopeptides. The glycopeptides were then treated with peptide:N-glycosidase F (PNGase F) and/or endo-beta-N-acetylglucosaminidase (Endo H) and the molecular mass of the deglycosylated peptide was determined by MALDI/MS and matched with the peptide predicted by a computer program. The sequences of some peptides or deglycosylated peptides were identified by the MALDI post-source decay technique. The size of the oligosaccharide, the degree of glycosylation and the distribution of the oligosaccharides at each individual potential glycosylation site were characterized. This information goes for beyond previously published data and sometimes differs from them. During this study, the amino acid sequence originally derived from the DNA sequence of the gene coding for invertase was also verified and it was found that this protein when expressed from SUC2 gene might be created as more than one sequence which differ by a few amino acid substitutions (Asn58<-->Thr, Asn65-->His and Val412<-->Ala).
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PMID:Determination of N-linked glycosylation of yeast external invertase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 1022 60

This review covers the unique catalytic and molecular properties of three proteolytic enzymes and a glycosidase from Aspergillus. An aspartic proteinase from A. saitoi, aspergillopepsin I (EC 3.4.23.18), favors hydrophobic amino acids at P1 and P'1 like gastric pepsin. However, aspergillopepsin I accommodates a Lys residue at P1, which leads to activation of trypsinogens like duodenum enteropeptidase. Substitution of Asp76 to Ser or Thr and deletion of Ser78, corresponding to the mammalian aspartic proteinases, cathepsin D and pepsin, caused drastic decreases in the activities towards substrates containing a basic amino acid residue at 1. In addition, the double mutant T77D/G78(S)G79 of porcine pepsin was able to activate bovine trypsinogen to trypsin by the selective cleavage of the K6-I7 bond of trypsinogen. Deuterolysin (EC 3.4.24.39) from A. oryzae, which contains 1g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. It was concluded that His128, His132, and Asp164 provide the Zn2+ ligands of the enzyme according to a 65Zn binding assay. Deuterolysin is a member of a family of metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH + D" motif and an aspartic acid as the third zinc ligand. Acid carboxypeptidase (EC 3.4.16.1) from A. saitoi is a glycoprotein that contains both N- and O-linked sugar chains. Site-directed mutagenesis of the cpdS, cDNA encoding A. saitoi carboxypeptidase, was cloned and expressed. A. saitoi carboxypeptidase indicated that Ser153, Asp357, and His436 residues were essential for the enzymic catalysis. The N-glycanase released high-mannose type oligosaccharides that were separated on HPLC. Two, which had unique structures of Man10 GlcNAc2 and Man11GlcNAc2, were characterized. An acidic 1,2-alpha-mannosidase (EC 3.2.1.113) was isolated from the culture of A. saitoi. A highly efficient overexpression system of 1,2-alpha-mannosidase fusion gene (f-msdS) in A. oryzae was made. A yeast mutant capable of producing Man5GlcNAc2 human-compatible sugar chains on glycoproteins was constructed. An expression vector for 1,2-alpha-mannosidase with the "HDEL" endoplasmic reticulum retention/retrieval tag was designed and expressed in Saccharomyces cerevisiae. The first report of production of human-compatible high mannose-type (Man5GlcNAc2) sugar chains in S. cerevisiae was described.
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PMID:Unique catalytic and molecular properties of hydrolases from Aspergillus used in Japanese bioindustries. 1083 Apr 77

A serine proteinase with kallikrein-like activity (LV-Ka) has been purified to homogeneity from bushmaster snake (Lachesis muta muta) venom. Physicochemical studies indicated that LV-Ka is a single chain glycoprotein with a molecular mass (Mr) of 33 kDa under reducing conditions which was reduced to 28 kDa after treatment with N-Glycosidase F (PNGase F). LV-Ka can be bounded and neutralized by serum alpha2-macroglobulin (alpha2-M), a prevalent mammalian protease inhibitor that is capable of forming a macromolecular complex with LV-Ka (Mr >180 kDa). Cleavage of alpha2-M by the enzyme resulted in the formation of 90-kDa fragments. The proteolytic activity of LV-Ka against dimethylcasein could be inhibited by alpha2-M, and the binding ratio of the inhibitor:enzyme complex was found to be 1:1. The Michaelis constant, Km, and catalytic rate constant, kcat, of LV-Ka on four selective chromogenic substrates were obtained from Lineweaver-Burk plots. LV-Ka exhibits substrate specificities not only for the glandular kallikrein H-D-Val-Leu-Arg-pNA (S-2266) but also for the plasmin substrates S-2251 and Tos-Gly-Pro-Lys-pNA. Bovine kininogen incubated with LV-Ka generated a polypeptide that dose dependently contracted mesenteric arterial rings from spontaneously hypertensive rats (SHR) in a similar way as bradykinin (BK) does. As it happens with BK, LV-Ka generated polypeptide was inhibited by HOE-140, a bradykinin B2-receptor antagonist and by indomethacin, a cyclo-oxygenase inhibitor. These results strongly suggest that the polypeptide generated by LV-Ka by cleavage of bovine kininogen is bradykinin. In addition, our studies may help to understand the mechanism of action involved in hypotension produced by envenomation of bushmaster snake.
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PMID:Biochemical properties of a bushmaster snake venom serine proteinase (LV-Ka), and its kinin releasing activity evaluated in rat mesenteric arterial rings. 1553 59

Dentin sialophosphoprotein (DSPP) is a major secretory product of odontoblasts and is critical for proper dentin formation. DSPP is believed to be processed into only two structural/functional domains: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Here we report the isolation and characterization of a third domain of DSPP, designated dentin glycoprotein (DGP). DGP was isolated from a guanidine/EDTA extract of porcine tooth dentin by ion exchange, hydroxyapatite affinity, size exclusion, and RP-HPL chromatography. Endoproteinase lysine C digestion products of DGP were characterized by Edman sequencing and mass spectrometry. The porcine DGP backbone is the 81-amino acid segment of DSPP (Ser392 to Gly472) between the DSP and DPP domains. DGP has four phosphorylated serine residues (Ser453, Ser455, Ser457, and Ser462) and one glycosylated asparagine (Asn397). There are no other post-translational modifications. DGP is a stains-all positive protein with an apparent molecular mass on SDS-PAGE of 19 kDa, which is reduced by glycopeptidase A digestion to 16 kDa. A variety of glycans can be linked to Asn397. All are complex biantennary structures with a common N-linked pentasaccharide core (mannose3-N-acetylglucosamine2), most with a fucosyl residue on the innermost N-acetylglucosamine. The alpha1-3 and alpha1-6 arms are always galactose beta1-4 N-acetylglucosamine beta1-2 mannose, and either or both arms can be unsialidated or monosialidated. The calculated monoisotopic molecular masses of the different glycosylated forms of the DGP phosphoprotein are: unsialidated 10,523 and 10,670, monosialidated 10,815 and 10,961, and disialidated 11,106, and 11,252 Da, with the disialidated forms being the most abundant.
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PMID:Dentin glycoprotein: the protein in the middle of the dentin sialophosphoprotein chimera. 1572 77

DPL2 (DPP10) found at chromosome 2q14.1 is a member of the dipeptidyl peptidase IV (DPIV) gene family. Here we characterize a novel short DPL2 isoform (DPL2-s), a 789-amino acid protein, that differs from the previously described long DPL2 isoform (DPL2-l) at the N-terminal cytoplasmic domain by 13 amino acids. The two DPL2 isoforms use alternate first exons. DPL2 mRNA was expressed mainly in the brain and pancreas. Multiple forms of recombinant DPL2-s protein were observed in 293T cells, having mobilities 96 kDa, 100 kDa, and approximately 250 kDa which may represent soluble DPL2, transmembrane DPL2 and multimeric DPL2 respectively. DPL2 is glycosylated as a band shift is observed following PNGase F deglycosylation. DPL2-s was expressed primarily on the cell surface of transfected 293T and PC12 cells. DPL2-s exhibits high sequence homology with other DPIV peptidases, but lacks a catalytic serine residue and lacks dipeptidyl peptidase activity. Substitutions of Gly(644)-->Ser, Lys(643)Gly(644)-->TrpSer, or Asp(561)Lys(643)Gly(644)-->TyrTrpSer in the catalytic motif did not confer dipeptidyl peptidase activity upon DPL2-s. Thus, although DPL2 is similar in structure and sequence to the other dipeptidyl peptidases, it lacks vital residues required to confer dipeptidyl peptidase activity and has instead evolved features that enable it to act as an important component of voltage-gated potassium channels.
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PMID:Molecular characterization of a novel dipeptidyl peptidase like 2-short form (DPL2-s) that is highly expressed in the brain and lacks dipeptidyl peptidase activity. 1629 Feb 53

Lysine-containing peptides comprising glycosylation sites derived from recombinant human erythropoietin (rHuEPO) by trypsin or Lys-C and PNGase F dual digestion were derivatized with 2-methoxy-4,5-dihydro-1H-imidazole and its deuterated analogues. In the same reaction, under reducing conditions (beta-mercaptoethanol), cysteines were converted into methyl-cysteines and lysines into Lys-4,5-dihydro-1H-imidazole. Both modifications on cysteines and lysines simplified the CID-MS/MS spectra, while preserving the structural information by yielding y-series ions and improved the mass spectral signal intensity up to 25 times. Moreover, by this approach, the N-glycan occupation sites were unambiguously determined. O-Glycosylation sites as well as O-glycan structures were determined by a LC-MS/MS experiment carried out on dually digested rHuEPO. N-Glycan mixture purified on a graphitized carbon column using a newly developed method that extracted only sialylated carbohydrates was analyzed first using MALDI-TOF in negative linear ion mode with low mass accuracy but without interferences and metastabile ions and then a reflectron with high mass accuracy. After defining the precursor ions, we performed the nanoESI QTOF MS/MS analysis on N-glycans, mainly targeting the distinction between carbohydrates with sialylated antennae and those lacking sialic acid moieties.
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PMID:Mass spectrometry-based glycoproteomic approach involving lysine derivatization for structural characterization of recombinant human erythropoietin. 1708 Oct 58

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.
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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

High-performance liquid chromatography (LC) and liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-MS) methods with various sample preparation schemes were compared for their ability to identify and quantify glycoforms in two different production lots of a recombinant monoclonal IgG1 antibody. IgG1s contain a conserved N-glycosylation site in the fragment crystallizable (Fc) subunit. Six methods were compared: (1) LC/ESI-MS analysis of intact IgG, (2) LC/ESI-MS analysis of the Fc fragment produced by limited proteolysis with Lys-C, (3) LC/ESI-MS analysis of the IgG heavy chain produced by reduction, (4) LC/ESI-MS analysis of Fc/2 fragment produced by limited proteolysis and reduction, (5) LC/MS analysis of the glycosylated tryptic fragment (293EEQYNSTYR301) using extracted ion chromatograms, and (6) normal phase HPLC analysis of N-glycans cleaved from the IgG using PNGase F. The results suggest that MS quantitation based on the analysis of Fc/2 (4) is accurate and gives results that are comparable to normal phase HPLC analysis of N-glycans (6).
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PMID:Comparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs. 1870

The human serotonin transporter (hSERT) is responsible for reuptake of serotonin (5-HT) from the synaptic cleft and is target for antidepressant medicine. Differential hSERT activity caused by genetic polymorphisms is believed to affect the risk of developing depression and, moreover, to affect the response to antidepressant therapy. The hSERT contains in the second extracellular loop (EL2) two sites for N-linked glycosylation that are critical for functional transporter expression. Here we examine a non-synonymous single nucleotide polymorphism (SNP) in EL2 that gives rise to a potential third glycosylation site due to substitution of a lysine at position 201 with an asparagine (K201N). In agreement with introduction of an additional glycosylation site, western blot analysis showed migration of hSERT K201N corresponding to a higher molecular weight than wild type hSERT upon expression in both HEK293 cells and primary cultures of cortical neurons. An increase in molecular weight was not observed after removal of glycans with peptide N-glycosidase F (PNGase F). Quantitative analysis of western blots indicated significantly increased total transporter expression ( approximately 30%) for hSERT K201N as compared to hSERT in both cell systems. The increase in expression was accompanied by corresponding significant increases in the number of [(3)H]citalopram binding sites and in the V(max) for [(3)H]5-HT uptake. Characterization of mutants carrying all possible combinations of glycosylation sites demonstrated clear correlation between the number of glycosylation sites and the level of transporter activity, and showed that K201N could substitute for either one of the two original glycosylation sites.
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PMID:A single nucleotide polymorphism in the human serotonin transporter introduces a new site for N-linked glycosylation. 1950 Jun 2

The principal objective of this study was the evaluation of two-dimensional gel electrophoresis (2-DE) in combination with MALDI-TOF MS, after tryptic digest with regard to suitability for qualitative characterization and identification of therapeutic recombinant monoclonal antibodies trastuzumab and rituximab. Moreover, the impact of post-translational modifications of these glycoproteins on the electrophoresis behavior has been evaluated. 1-D SDS-PAGE, in reducing and non-reducing conditions, and 2-DE were used for the assessment of M(r) and the monitorization of deglycosylation efficiency. In addition, 2-DE was used for the determination of pIs. 2-DE gels revealed characteristic glycoprotein migration behavior, highly complex spot pattern, typical for recombinant monoclonal antibodies. N-linked oligosaccharides were released with PNGase F; enzymatic desialination was studied with sialidase and carboxypeptidase B was used for the study of lysine truncation. Peptide spots resolved in 2-DE gels were in gel tryptically digested, resulting peptides were subjected to MALDI-TOF MS analysis and peptide mass fingerprinting (PMF) has been used for the identity confirmation of both monoclonal antibodies.
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PMID:Comparison of two-dimensional gel electrophoresis patterns and MALDI-TOF MS analysis of therapeutic recombinant monoclonal antibodies trastuzumab and rituximab. 2181 59


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