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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 effect of carbohydrate removal on the properties of the lysosomal enzyme alpha-L-fucosidase has been investigated by comparatively characterizing N-glycanase-treated and mock-treated control fucosidases. N-Glycanase treatment removed approx. 90% of the carbohydrate from purified native human liver fucosidase as determined by carbohydrate assay after gel filtration on Sephadex G-50, and by Western blotting with a lectin-digoxigenin conjugate and densitometric scanning. Removal of carbohydrate from fucosidase does not affect its catalytic activity, its Km value for synthetic substrate, its recognition and rate of hydrolysis of three natural substrates, or its gross conformation as determined by circular dichroism. However, loss of carbohydrate led to significantly decreased activity at acidic pH values (3.1-4.7), a 0.6 pH unit shift to a more neutral optimum and decreased thermostability. The decreased activity at acidic pH values and the more neutral pH optimum of deglycosylated fucosidase suggest that the presence of carbohydrate is physiologically significant in allowing fucosidase to perform its catabolic function more efficiently in the acidic milieu of the lysosome.
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PMID:The effect of carbohydrate removal on the properties of human liver alpha-L-fucosidase. 154 Jun 52

The thrombin-like serine protease and antithrombotic agent, Ancrod, was rapidly purified from the crude venom of Akistrodon rhodostoma by agmatine-Sepharose affinity chromatography followed by MonoQ anion exchange chromatography. N-Terminal sequencing and analysis of overlapping proteolytic fragments of purified Ancrod by automated Edman degradation in combination with tandem mass spectroscopy allowed the determination of the 234 amino acid sequence of the protease. Glycosylation sites at all five canonical N-linked glycosylation sites were inferred from the appearance of blank sequencer cycles in the amino acid sequence and were confirmed by mass spectroscopic analysis of the N-glycanase-treated peptides. Monoclonal antibodies raised against the denatured protein and HF-deglycosylated protein recognized Ancrod on Western blots. Sequence comparison to other thrombin-like serine proteases and reptilian fibrinogenases revealed a number of similarities, most notably the catalytic triad and many conserved cysteine positions.
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PMID:Amino acid sequence determination of ancrod, the thrombin-like alpha-fibrinogenase from the venom of Akistrodon rhodostoma. 154 12

Previous studies have established the importance of a complex, N-linked oligosaccharide chain, recognized by a monoclonal antibody (mAb 1223), in the formation of sea urchin embryonic skeletal components known as spicules. To further investigate the function of this epitope, mAb 1223 was added to primary mesenchyme (PM) cell cultures prior to spiculogenesis. The antibody did not inhibit cell migration, cell attachment, or synthesis of the filapodial networks upon which the spicules are deposited. However, it did block deposition of mineralized CaCO3 along these filapodia, strongly supporting the previously proposed role for the 1223 epitope in calcium accumulation and/or deposition. Previously the 1223 epitope has been most extensively studied in association with a mesenchyme-specific protein of 130 kDa (msp 130). It has now been established, by Western blot analysis of whole embryo and PM cell extracts using mAb 1223, that two other proteins of 205 and 250 kDa contain the 1223 epitope. A study of the developmental profiles of expression of these glycoproteins revealed that all three were first expressed just prior to spiculogenesis, consistent with a role for any or all of these proteins in this process. Additionally all three proteins incorporated ethanolamine, myristate, and palmitate, the precursors of the glycosylphosphatidylinositol (GPI) anchor. Further labeling studies revealed differences in the metabolic lability of the GPI anchor in the three proteins; pulse-chase studies demonstrated that the ethanolamine moiety was stable in msp 130, but was rapidly chased from the 205-kDa protein (T1/2 = 14 hr). Phosphatidylinositol-specific phospholipase C partially released (50%) msp 130 from the PM cell surface, whereas it had no effect on release of the 205- and 250-kDa proteins. Studies with 35SO4 labeling and PNGase F treatment directly established that all three proteins are sulfated, and that most of the sulfate is attached to the N-linked oligosaccharide chains. Thus, the three major mAb 1223-reactive glycoproteins in PM cells are also the three major proteins containing both sulfated N-linked oligosaccharide chains and GPI anchors. Further investigation of this intriguing correlation may help to define the precise function of the 1223 epitope in the process of spicule formation.
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PMID:Characterization of post-translational modifications common to three primary mesenchyme cell-specific glycoproteins involved in sea urchin embryonic skeleton formation. 155 76

Human transferrin receptor was isolated from placenta and from the hepatocarcinoma cell line Hep G2. Asparagine-linked oligosaccharides were released by treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Oligosaccharide alditols were fractionated by anion-exchange high-performance liquid chromatography and by high-pH anion-exchange chromatography. Glycans from placental transferrin receptor were further characterized, after desialylation, by methylation analysis and, in part, by liquid secondary-ion mass spectrometry. Sialylation of placental transferrin receptor was examined by lectin affinity blotting with Sambucus nigra agglutinin and Maackia amurensis agglutinin. In order to trace possible inter-individual differences in N-glycosylation of the receptor, two preparations of placental transferrin receptor purified from two donors were compared. The results demonstrate that human transferrin receptor from placenta predominantly carries diantennary and triantennary N-acetyllactosaminic glycans as well as hybrid-type species, the galactose residues of which being almost completely substituted with (alpha 2-3)-linked sialic acid residues. Distinct differences were noted in the glycosylation pattern of the receptor from different individuals. Transferrin receptor from donor A carried predominantly diantennary and triantennary complex-type glycans, in part fucosylated at the innermost N-acetylglucosamine residue, in addition to small amounts of bisected and of incomplete diantennary species. Placental transferrin receptor from donor B predominantly carried triantennary N-acetyllactosaminic glycans without fucose and hybrid-type oligosaccharides with four or five mannose residues. Distinct from placental transferrin receptor, the receptor from Hep G2 cells contained larger amounts of oligomannosidic glycans with six to nine mannose residues and tetrasialylated complex-type oligosaccharides apart from mono-, di- and trisialylated species.
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PMID:Structure of the N-linked oligosaccharides of the human transferrin receptor. 155 86

Two receptor polypeptides have been identified in several studies by using cross-linking with interleukin 3 (IL-3). It has been suggested that proteolytic degradation of the larger polypeptide yields the lower molecular weight fragment, but there is little proof that this is the case. We have used several different approaches to characterize the polypeptides cross-linked in R6X or FDC-P1 cells. Several bifunctional cross-linkers of various sizes were tested to determine their effectiveness in cross-linking IL-3 to its receptor. The longer cross-linker gave the highest proportion of labeling of the low molecular weight band. Incubation in the absence of protease inhibitors caused a decrease in labeling of both cross-linked polypeptides, but no indication of a significant increase in the lower molecular weight band. Direct comparison of the two cross-linked polypeptides by V8 protease mapping showed no common peptides that might be expected if they were related molecules, except those derived from the iodinated IL-3. Digestion with N-glycanase resulted in a decrease in apparent molecular weight of 5000 in the larger polypeptide but a decrease of 15,000 in the smaller polypeptide. These results suggest that the 70-kd polypeptide identified by cross-linking of IL-3 represents a second binding chain of the receptor. By analogy with some of the other hemopoietin receptors, the 70- and 125-kd polypeptides may form a complex necessary for high affinity binding.
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PMID:Two polypeptides identified by interleukin 3 cross-linking represent distinct components of the interleukin 3 receptor. 156 67

The production of TNF-alpha and TNF-beta by human B-cell lines was studied at both the molecular and biological levels. The 24 B-cell lines studied included EBV+ cell lines (n = 13), EBV- cell lines (n = 8), and AIDS-associated B-cell lines (AABCL) (n = 3) which are EBV+/HIV-. Whereas radioimmunoprecipitation using TNF-alpha antisera detected 17-kDa TNF-alpha as expected, similar studies with anti-TNF-beta antisera revealed TNF-beta microheterogeneity. In the AABCL three bands with approximate MW of 26, 24, and 22 kDa were detected under reducing conditions, and in the non-AABCL, two bands only with 26 and 22 kDa were observed. To determine whether the size heterogeneity of TNF-beta is due to glycosylation, TNF-beta deglycosylation studies were done in two AABCL (PA682BM-2, PA682PE-1) and one non-AABCL (IM-1178). As control, the normal lymphoblastoid B-cell line RPMI-1788, which is known to secrete TNF-beta with MW 25 and 20 kDa, has been used. Deglycosylation studies using N-glycanase + neuraminidase + O-glycanase reduced the various bands in all cell lines to one band with 18.6 kDa, which is compatible with the TNF-beta backbone. In attempt to determine whether the differential glycosylation of TNF has any functional significance, all 24 cell lines were studied for TNF secretion and for TNF neutralization by monoclonal antibodies and polyclonal antibodies to TNF-alpha and TNF-beta. Constitutive secretion of TNF-alpha and TNF-beta has been detected only in the three AABCL. Following activation with the tumor promoter teleocidin, the secretion of both TNFs has been triggered in 2/8 EBV- cell lines and in 8/13 EBV+ non-AABCL. Using rabbit polyclonal antibodies to human TNF-alpha and to human TNF-beta, only little if any neutralization of these TNFs has been shown. Our data suggest that the differences in glycosylation of B-cell-derived TNFs may account for the incomplete neutralization, and may influence the cytotoxic biological activity of this lymphokine.
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PMID:Human B-cell TNF-beta microheterogeneity. 157 46

Urea-soluble fractions from purified Kurloff cells (KC) were analysed by affinoblotting. Lectin reactivities were quasi-exclusively confined to the 30-35 kDa major glycoproteins (mGPs) (responsible for the PAS positivity of the Kurloff body) with strong affinities for Canavalia ensiformis lectin, Phaseolus vulgaris erythroagglutinin and Sambucus nigra (SNA), Pisum sativum, Triticum vulgaris and Ulex europeus agglutinins. These data were consistent with the presence, among the KC mGPs, of large amounts of complex or hybrid N-glycosylproteins, in particular with Neu5Ac alpha 2,6Gal/GalNAc sequences, fucosyl residues and bisected residues. Their oligosaccharide sequences belong to more than one class, since some of these lectin reactivities had to be borne by distinct N-linked oligosaccharide chains. Before further analysis, KC mGPs were separated from other highly anionic glycoconjugates, by DEAE-cellulose chromatography. Their abundant potential RCA-binding sites masked by sialic acid were then revealed after neuraminidase (sialidase) or dilute acid pre-treatment. In remaining consistent with their lectin affinities, some KC mGPs were found to be PNGase F sensitive, while, either desialylated or not, they were all O-glycanase insensitive. Finally, by combined zymography and affinoblotting, the SNA-reactive fraction of KC mGPs was shown to correspond to denatured forms of the two zymographic size populations (190 kDa and 500 kDa) of KC acid phosphatases.
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PMID:The major Kurloff cell glycoproteins: lectin affinities, glycosidase susceptibilities and relationship with the sialylated acid phosphatases of the Kurloff body. 158 39

Two glycoproteins were isolated from lysates of thioglycollate-stimulated, murine peritoneal macrophages by affinity chromatography on immobilized Griffonia simplicifolia I lectin and by preparative SDS/PAGE. The glycoproteins were readily labeled on the surface of intact macrophages with 3H and 125I. The labeled glycoproteins migrated as broad bands of molecular mass 92-109 kDa and 115-125 kDa. The mobility of the glycoproteins decreased only slightly after reduction with dithiothreitol, indicating the absence of intersubunit disulfide bridges. The 92-kDa and 115-kDa glycoproteins had pI 5.2-5.4 and pI less than or equal to 4, respectively. Digestion of both glycoproteins with alpha-galactosidase released 23% of their 3H content and abolished their ability to bind to the G. simplicifolia I lectin, showing that they contain terminal alpha-D-galactosyl groups. After reduction with 2-mercaptoethanol, each glycoprotein fraction was sensitive to N-glycanase; the 115-kDa glycoproteins produced a smear with the front at approximately 67 kDa, whereas the 92-kDa glycoprotein gave two bands of 61 kDa and 75 kDa. Unreduced glycoproteins were insensitive to N-glycanase, suggesting the presence of intramolecular disulfide bonds. Although each glycoprotein fraction was sensitive to endoglycosidase H, this enzyme produced only slight changes in molecular mass when compared with N-glycanase. From these results as well as from the specificity of the enzymes involved, it is concluded that each glycoprotein fraction contains complex-type oligosaccharides and a small amount of high-mannose and/or hybrid-type oligosaccharides. While each glycoprotein fraction was bound to Datura stramonium lectin, they failed to react with anti-[i-(Den)] serum and their digestion with endo-beta-galactosidase did not cause a band shift in SDS/PAGE. Taken together, these results suggest the presence of N-acetyllactosamine units which are not arrayed in linear form but occur as single units, bound either to C2 and C6, or to C2 and C4, or both, of outer mannosyl residues on complex-type oligosaccharides. The glycoprotein(s) fraction precipitated with anti-[I (Step)] serum, suggesting the presence of branched lactosaminoglycans. Digestion of both glycoprotein fractions with a mixture of sialidase and O-glycanase did not alter their mobility in SDS/PAGE, suggesting a lack or low content of O-linked trisaccharides and tetrasaccharides. Each glycoprotein fraction was bound specifically to Sambucus nigra and Maackia amurensis immobilized lectins, indicating the presence of sialic acid linked alpha 2,6 to subterminal D-galactose or N-acetylgalactosamine residues, and alpha 2,3 to N-acetyllactosamine residues, respectively.
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PMID:alpha-D-galactose-bearing glycoproteins on the surface of stimulated murine peritoneal macrophages. Biochemical and immunochemical characterization of purified glycoproteins. 158 69

Zona pellucida (ZP), the extracellular glycocalyx surrounding the mammalian oocyte, is believed to mediate species-specific sperm-egg interaction. Despite numerous studies on characterization of ZP glycoconjugates in several species, little or no information is available on the number and chemical nature of the various components of the rat ZP. In this study we have attempted the biochemical characterization of the rat ZP using endo- and/or exo-glycohydrolases. Intact eggs from superovulated rats were radioiodinated by the chloramine-T method, and the labeled ZP components were resolved on SDS-PAGE under nonreducing conditions. These studies show that the rat ZP consists of three components with apparent molecular masses of 205 kDa (ZP1), 119 kDa (ZP2), and 115 kDa (ZP3). Unlike mouse ZP2 and ZP3, which resolve as distinct components on SDS-PAGE, rat ZP2 and ZP3 show substantial overlap in their molecular sizes and isoelectric points. Treatment of the rat ZP components with exo- (neuraminidase and alpha-L-fucosidase) and/or endo- (endoglycosidase H, endoglycosidase F, N-glycanase, and O-glycanase) glycohydrolases indicated the following: 1) Both rat ZP2 and ZP3 contain N-linked oligosaccharide (OS) units as indicated by their sensitivity to endoglycosidase F and N-glycanase. 2) Treatment with N-glycanase caused a reduction in size of the rat ZP2 and ZP3 components by nearly 50% and 60%, respectively, indicating that the two ZP components are highly glycosylated. 3) Rat ZP3 was sensitive to O-glycanase, suggesting that this ZP component contains O-linked OS unit(s). 4) No evidence was obtained for the presence of fucosyl or sialyl residue(s) on the O-linked OS unit(s) of rat ZP3.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Qualitative characterization of oligosaccharide chains present on the rat zona pellucida glycoconjugates. 159 46

Characteristic properties of the antigens recognized by sperm-immobilizing monoclonal antibodies (SI-mAbs) from different sources were compared by ELISA competitive inhibition assay, Western blot analysis, chromatographic analysis, and enzymatic digestion studies. Among 9 SI-mAbs, human mAb H6-3C4 and three mouse mAbs--2C6, 2B6, and 2E5--also possessed strong sperm-agglutinating activity. Binding of human mAb H6-3C4 to sperm was strongly inhibited by the three mouse mAbs (2C6, 2B6, and 2E5), but not by the rat or the other four mouse mAbs. SDS-PAGE revealed that mAb H6-3C4 and three mouse mAbs recognized the same antigen molecules of 15-25 kDa present in both sperm extracts and seminal plasma. Chemical treatments with trifluoromethanesulfonic acid and sodium metaperiodate destroyed the antigen determinants recognized by the above four mAbs, as detected by both ELISA and antibody absorption tests. Western blot analysis revealed that the antigens were susceptible to treatments with papain, proteinase K, and N-glycanase, but resistant to trypsin, V8 protease, and thermolysin. These results indicate that one of the major antigens recognized by mAbs with sperm-immobilizing action may be a sperm membrane-associated glycoprotein of 15-25 kDa and the epitope may involve N-linked oligosaccharides.
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PMID:Comparative studies of the antigens recognized by sperm-immobilizing monoclonal antibodies. 161 9


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