Substrate specificity of Flavobacterium meningosepticum Endo F2 and endo F3: purity is the name of the game.
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Amino Acid Sequence↗
Biomedical subjects
T H Plummer
Publications and source records attributed to T H Plummer.
The first demonstration of a procaryotic glycosylasparaginase.
Glycosylasparaginase was purified to near homogeneity from intracellular lysates of Flavobacterium meningosepticum. The enzyme is a heterodimer with an estimated molecular weight of 38 kDa and consists of one alpha-subunit (18 kDa) and one beta-subunit (16 kDa). The beta-subunit of the Flavobacterium enzyme has a direct evolutionary relationship to the beta-subunit of mammalian glycosylasparaginases as evidenced by: (1) strong cross-reactivity with antibodies made to the denatured rat beta-subunit, (2) a high degree of homology with the amino-terminus of the corresponding eukaryotic enzymes, and (3) irreversible inactivation with 5-diazo-4-oxo-L-norvaline, a reagent known to react with the catalytic amino-terminal threonine residue on the beta-subunit of a mammalian glycosylasparaginase.
Amidohydrolases↗
Multiple endoglycosidase F activities expressed by Flavobacterium meningosepticum endoglycosidases F2 and F3. Molecular cloning, primary sequence, and enzyme expression.
The genes for Flavobacterium meningosepticum Endo (endoglycosidase) F2 and Endo F3 were cloned, and their nucleotide sequences were determined. The deduced amino acid sequences were verified independently to a large extent by direct peptide microsequencing of 66 and 84% of native Endo F2 and Endo F3, respectively. Structurally, the Endo F2 and Endo F3 genes code for a typically long leader sequence of 45 and 39 amino acids, respectively, and, in both cases, a mature protein of 290 amino acids. Comparative structural analysis demonstrated minimum overall homology (15-30%) between Endo F1, Endo F2, and Endo F3, but revealed distinct clusters of identical residues distributed throughout the entire sequence, which represent motifs for binding and hydrolysis of beta 1,4-di-N-acetylchitobiosyl linkages in complex carbohydrates. The mobility of native Endo F2 and Endo F3 on SDS-polyacrylamide gel electrophoresis, unlike Endo F1, did not correlate with the molecular weights determined from the coding region of the corresponding genes. Mass spectrometry confirmed that Endo F2 and Endo F3 were heterogeneous and contained approximately 4000 and 1200 daltons of mass not accounted for in the gene structure. We presume that Endo F2 and Endo F3 are variably post-translationally modified during secretion by possible linkage to the hydroxyl of serine.
Amino Acid Sequence↗
2-Iminothiolane: a reagent for the introduction of sulphydryl groups into oligosaccharides derived from asparagine-linked glycans.
A facile method for introducing reactive sulphydryl groups into oligosaccharides was developed. 1-Amino-oligosaccharides generated from asparagine-linked glycans by peptide-N4(N-acetyl-beta-D-glucosaminyl) asparagine amidase (PNGase F) digestion were monitored by high-performance anion-exchange chromatography with pulsed amperometric detection and derivatized under optimal conditions with 2-iminothiolane-HCl. The resulting mercapto-butyramido oligosaccharides, which were obtained in high yield, were alkylated with a fluorescent reagent and used to selectively assay for endoglycosidases that hydrolyse di-N-acetylchitobiose linkages.
Asparagine↗
Multiple endoglycosidase (Endo) F activities expressed by Flavobacterium meningosepticum. Endo F1: molecular cloning, primary sequence, and structural relationship to Endo H.
A full-length insert for the endo-beta-N-acetylglucosaminidase (Endo) F1 gene was located on a 2,200-base pair EcoRI fragment of genomic DNA and cloned into the plasmid vector Bluescript. Transformed Escherichia coli cells expressed Endo F1 activity very well, but the enzyme apparently was not processed and secreted into the medium as it normally is in Flavobacterium meningosepticum. DNA sequencing revealed an open reading frame of 1,017 nucleotides encoding a putative 50-amino acid signal sequence, and a mature protein (31,667 Da) of 289 amino acids. The deduced amino acid sequence was verified by direct Edman microsequencing of 88% of the purified protein as tryptic and V8 protease peptides. Alignment of Endo F1 (289 amino acids) with the established amino acid sequence of Streptomyces plicatus Endo H (271 amino acids) revealed a 32% structural identity over the entire sequence and a high degree of conservative replacements. Potential catalytic domains identified in other proteins that hydrolyze the beta 1,4 glycosidic linkage between N-acetylglucosamine residues are also conserved for amino acid identity and relative spacing in Endo F1.
Amino Acid Sequence↗
Purification of the oligosaccharide-cleaving enzymes of Flavobacterium meningosepticum.
Four oligosaccharide chain-cleaving enzymes, including two new endoglycosidases distinct from endo-beta-acetylglucosaminidase (Endo) F1, have been identified and purified to homogeneity from cultural filtrates of Flavobacterium meningosepticum. FPLC-directed hydrophobic-interaction chromatography in conjunction with high-resolution ion-exchange chromatography provided a more simple, rapid method for the isolation of endoglycosidase F1, F2 and F3, and the amidase, peptide-N4-N-acetyl-beta-D-glucosaminyl)-asparagine amidase (PNGase F), in greater than 50% yield. The specificity of PNGase F and Endo F1 are well established. Endo F2 and Endo F3 represent new distinct endoglycosidases that prefer complex as compared to high-mannose asparagine-linked glycans. Endo F2 cleaved biantennary oligosaccharides, whereas Endo F3 cleaved both bi- and triantennary oligosaccharides.
Chromatography, Gel↗
Molecular cloning and amino acid sequence of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase from flavobacterium meningosepticum.
A 3,000-base pair EcoRI fragment containing the Flavobacterium meningosepticum gene for peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase was cloned into the Bluescript plasmid vector and expressed in Escherichia coli. The gene consists of an open reading frame of 1,062 base pairs coding for a 354-amino acid protein; the first 40 amino acids are presumed to be the natural secretory signal sequence, with the remaining 314 amino acids (34,779 Da) representing the catalytically active protein. The deduced amino acid sequence was verified independently by direct microsequencing of over 94% of the pure protein (Flavobacterium peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase) as tryptic and cyanogen bromide peptides. Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase was not secreted by E. coli; molecular weight analysis of the partially purified recombinant enzyme suggested incomplete processing of the putative leader sequence.
Amidohydrolases↗
Carboxypeptidase H. A regulatory peptide-processing enzyme produced by human hepatoma Hep G2 cells.
Human hepatoma (Hep G2) cells have been shown to secrete nanogram quantities of carboxypeptidase N (Grimwood, B. G., Plummer, T. H., Jr., and Tarentino, A. (1988) J. Biol. Chem. 263, 14397-14401). A second carboxypeptidase with an acidic pH optimum (pH 5.5) is also secreted at levels 2-3-fold greater than carboxypeptidase N. This enzyme was partially purified from the conditioned medium and compared with pure bovine pituitary carboxypeptidase H. The two enzymes behaved in a similar fashion in DE52 ion-exchange chromatography and on gel filtration, with the Hep G2 enzyme being slightly larger than the bovine pituitary enzyme (52-54 versus 50-52 kDa). Both enzymes hydrolyzed COOH-terminal basic amino acids from typical synthetic substrates as well as from natural leuenkephalin peptides and were identical based on pH activity profiles, inhibition by EDTA or guanidinoethyl mercaptosuccinic acid, and stimulation by Co2+ ions. Inhibition of enzyme secretion from Hep G2 cells by tunicamycin indicated that the Hep G2 enzyme was glycosylated. This finding was confirmed by a parallel deglycosylation of the Hep G2 and bovine pituitary carboxypeptidase H enzymes with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Immunoblots using mouse antiserum to bovine pituitary carboxypeptidase H revealed that the Hep G2 enzyme was immunocross-reactive with the bovine enzyme but was slightly larger in size (54 versus 52 kDa). Continuous [35S]methionine labeling and purification to near homogeneity using an affinity matrix corroborated the observations that the secreted Hep G2 carboxypeptidase H was slightly larger than bovine pituitary carboxypeptidase H. The Hep G2-secreted enzyme in pulse-chase experiments was initially detected intracellularly after a 15-min pulse as a single protein of about 54 kDa and was present in the 30-min chase medium with no evidence for pre- or postsecretion proteolytic processing. The human adrenergic cell line IMR-32 continuously labeled with [35S]methionine also secreted carboxypeptidase H of the same size as the Hep G2 enzyme.
Animals↗
Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases.
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Acetylglucosaminidase↗
Enzymatic approaches for studying the structure, synthesis, and processing of glycoproteins.
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Amidohydrolases↗
Characterization of the carboxypeptidase N secreted by Hep G2 cells.
Human hepatoma (Hep G2) cells secrete nanogram quantities of carboxypeptidase enzymes which are capable of hydrolyzing COOH-terminal lysine and arginine residues. A carboxypeptidase with a neutral pH optimum (greater than pH 7.0) was partially purified from the conditioned medium and compared with pure plasma carboxypeptidase N. The two enzymes behaved in a similar manner on gel filtration (apparent Mr = 280,000), DE52 ion exchange chromatography, and concanavalin A-affinity chromatography and were indistinguishable enzymatically and immunologically. Immunoblots of the Hep G2 and plasma carboxypeptidase N before and following deglycosylation with peptide-N4-[N-acetyl-beta-glucosaminyl]asparagine amidase F revealed a similar, if not identical, multimeric structure. A second carboxypeptidase with a lower molecular weight and a pH optimum of 5.0 was also detected in the Hep G2 medium.
Animals↗
High performance liquid chromatographic quantitation of carboxypeptidase activity secreted by human hep G2 cells.
A new high performance liquid chromatographic method has been developed for the determination of carboxypeptidase N activity which quantitates the furylacryloyl-alanine released by enzymatic cleavage of furylacryloyl-alanyl-lysine or furylacryloyl-alanyl-arginine. A short isocratic gradient elutes the substrate and product in less than 7 min and multiple analyses are facilitated by an automatic sample injector. The microassay readily detects and quantitates carboxypeptidase N activity secreted into culture medium. It was determined that approximately 1 X 10(6) Hep G2 cells at early confluence secreted 1 ng of carboxypeptidase N in 24 h. The microassay will also detect as little as 51 pg of purified carboxypeptidase N or 8 pg of carboxypeptidase B.
Carboxypeptidases↗
Structural characterization of intact, branched oligosaccharides by high performance liquid chromatography and liquid secondary ion mass spectrometry.
We report results of a mass-spectrometric-based strategy for determining the detailed structural features of N-linked oligosaccharides from glycoproteins. The method was used to characterize a series of intact, high mannose oligosaccharides isolated from human immunoglobulin M (IgM). The IgM was purified from a patient with Waldenstrom's macroglobulinemia. The strategy included releasing the oligosaccharides by digestion of the purified glycoprotein with endoglycosidase H, separating the released oligosaccharides by high resolution gel filtration, and derivatizing the resulting reducing termini with the uv-absorbing moiety, ethyl p-aminobenzoate. This particular derivative facilitates HPLC detection and provides centers for protonation and deprotonation enhancing liquid secondary ion mass spectra. Positive and negative ion spectra contained molecular species of similar abundance. However, fragment ion peaks yielding sequence information were significantly more prominent in the negative ion mass spectra. Furthermore, it was obvious that the fragmentation patterns differed substantially for linear and branched oligomers. For linear oligosaccharides, a smooth envelope of fragment ions was observed; from low to high mass there was an ordered decrease in ion abundance from both the reducing and nonreducing termini. This pattern of fragment ions was not observed for branched oligosaccharides since in these cases fragments at certain masses could not arise by single bond cleavages. Therefore, these fragments were either significantly reduced in abundance or absent as compared with identical fragments formed from linear molecules. Importantly, 200 pmol of an oligosaccharide could be derivatized, separated, and detected by mass spectrometry, allowing identification of previously unreported minor components of the IgM oligosaccharides. Therefore, this experimental strategy is particularly useful for the purification and detailed structural characterization of low abundance oligosaccharides isolated from heterogeneous biological samples.
Carbohydrate Conformation↗
Detection and quantification of peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidases.
A detailed study of the oligosaccharide specificity of the almond enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A, was undertaken by comparing the rate of release of intact oligosaccharide chains from defined glycopeptides of all significant classes. The oligosaccharide of a trisialo-triantennary pentaglycopeptide from fetuin was released at the highest rate. A procedure was developed for the isolation of this glycopeptide in high yield from 5 g fetuin. Sequence analysis established the structure as Leu-Ala-Asn(CHO)-Cys-Ser. The Cys(Cm) and the Cys(Ae) derivatives of the glycopeptide were reacted with 4-(dimethylamino)-azobenzene-4'-sulfonyl (dabsyl) chloride to yield a monosubstituted and a disubstituted glycopeptide respectively. This chromophore confers high sensitivity at 436 nm on a pentapeptide backbone having minimal bonds for protease cleavage. A procedure was developed wherein these dabsyl derivatives were used in a high-performance liquid chromatography assay. The dabsyl-pentapeptide was retarded significantly from the dabsyl-glycopeptide and provided a sensitive method (1-2 nmol) of detection of peptide-N4-(N-acetylglucosaminyl)asparagine amidase activity. Enzyme was detected in crude extracts of all eight seed sources surveyed. The enzyme from Pisum sativum was partially purified and its properties were compared with the corresponding enzyme from almonds.
Amidohydrolases↗
Peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase and endo-beta-N-acetylglucosaminidase from Flavobacterium meningosepticum.
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Acetylglucosaminidase↗
Conversion of MM creatine kinase isoforms in human plasma by carboxypeptidase N.
This study was undertaken to identify the carboxypeptidase(s) (CPase) in plasma mediating sequential conversion of the tissue isoform of the MM isoenzyme of creatine kinase (MM3 CK) to MM2 and MM1 isoforms and to elucidate relationships between CPase activity measured in plasma and observed rates of isoform conversion in vitro. Purified MM3 was incubated at 37 degrees C in plasma from normal subjects and patients with acute myocardial infarction. Isoforms were quantified by chromatofocusing. Preincubation with antiserum to CPase N prevented conversion of added MM3 to MM2 and MM1. Isoform conversion rates in the absence of antibody were proportional to plasma CPase N activity assayed spectrophotometrically by hydrolysis of furylacryloyl-L-alanyl-L-lysine substrate (r = 0.89, n = 8). Plasma CPase N activity varied by nearly 300% among individuals, but average activity was similar in samples from normal subjects (267 +/- 45 [SD] U/L, n = 18), those from outpatients with angina (289 +/- 43 U/L, n = 9), and those obtained at hospital admission from patients with acute infarction (Q wave: 279 +/- 70 U/L, n = 16; non-Q wave: 272 +/- 61 U/L, n = 14) or unstable angina (280 +/- 71 U/L, n = 11). In patients with Q wave infarction, CPase N activity increased by 43% +/- 25% between 48 hours and 72 hours (P less than 0.005 compared with admission) with a concomitant change in the rate of conversion of isoforms. Thus, the rate of conversion of isoforms in individual subjects can be estimated by assay of CPase N activity in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)
3-Mercaptopropionic Acid↗
Transfer of glycerol by Endo-beta-N-acetylglucosaminidase F to oligosaccharides during chitobiose core cleavage.
N-Linked oligosaccharides, when hydrolyzed by glycerol-containing preparations of endo-beta-N-acetylglucosaminidase (Endo) F from Flavobacterium meningosepticum were found to have glycerol attached to their reducing ends. The absence of a reducing end was confirmed by high-field 1H NMR spectroscopy, and the incorporated glycerol was verified through mass spectrometry and collisionally activated decomposition fast atom bombardment/mass spectrometry/mass spectrometry techniques. Periodate oxidation of [1(3)-14C]glycerol-labeled oligosaccharides indicated glycerol was glycosidically linked via its 1(3) carbon to the C1 of the reducing end N-acetylglucosamine. In a second, less favored reaction, the glycerol glycoside was hydrolyzed by Endo F using water as the terminal nucleophile, thus regenerating the N-acetylglucosamine reducing end. Glycerol could be removed from Endo F preparations without affecting enzyme stability, and chitobiosyl core hydrolysis in its absence provided intact oligosaccharides with normal N-acetylglucosamine reducing ends. The incorporation of labeled glycerol may provide a useful method for monitoring of Endo F release of oligosaccharides.
Acetylglucosamine↗
Inactivation of C3a by a monocarboxypeptidase present in culture supernatants of stimulated guinea pig peritoneal macrophages.
Hog C3a, as well as its derivative C3a-desArg were not found to act cytotoxically on starch gel-induced guinea pig peritoneal macrophages. Likewise, neither peptide significantly modified the secretion of N-acetyl-beta-D-glucosaminidase from these cells. However, C3a rapidly lost its spasmogenic activity during incubation in serum-free macrophage cultures and less rapidly in cellfree supernatants collected from cultured macrophages. The following results indicate that C3a is converted into its spasmogenically inactive derivative C3a-desArg by a macrophage-derived monocarboxypeptidase. The inactivated C3a product does not differ from native C3a in sodium dodecyl sulfate-polyacrylamide gel electrophoresis; it elutes from CM cellulose in the same position as purified C3a-desArg; and it is devoid of the carboxyl-terminal arginyl residue of C3a, but still contains the carboxyl-terminal sequence of C3a-desArg as determined by analysis after treatment with carboxypeptidases B or Y. Furthermore, inactivation of C3a in supernatants of macrophage cultures is completely blocked by the specific carboxypeptidase inhibitors guanidinopropylsuccinic acid and 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid in final concentrations of 10 mM and 2.1 mM, respectively. The monocarboxypeptidase is apparently supplied by biosynthesis of new material but is not stored as a preformed enzyme because cycloheximide markedly inhibits its expression.
Acetylglucosaminidase↗