Chemoenzymatic preparation of dermatan sulfate oligosaccharides as arylsulfatase B and alpha-L-iduronidase substrates.

Dermatan sulfate was partially depolymerized with chondroitin ABC lyase to obtain an oligosaccharide mixture from which an unsaturated disulfated tetrasaccharide was purified and characterized using nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. Chemical removal of the unsaturated uronate residue with mercuric acetate, followed by de-4-O-sulfation with arylsulfatase B (N-acetylgalactosamine 4-sulfatase) and N- acetylhexosaminidase catalyzed removal of the 2-acetamido-2-deoxy-D-galactospyranosyl residue at the non-reducing end afforded a monosulfated disaccharide of the structure alpha-L-idopyranosyluronic acid (1-->3)-alpha,beta-D-2-acetamido-2-deoxy-4-O-sulfo galactopyranose. This monosulfated disaccharide serves as a substrate for mammalian alpha-L-iduronidase as demonstrated using fluorophore assisted carbohydrate electrophoresis.

Direct measurement of unfractionated heparin using a biochemical assay.

A number of investigations have noted that functional biological assays for heparin are not always reliable and may not reflect the actual biochemical level of heparin in patients receiving anticoagulant therapy. This creates the possibility that patients receiving anticoagulant treatment may have an excess or deficiency of circulating levels of heparin. To address this problem, we have developed a direct biochemical measurement of heparin. The heparin assay uses fluorophore-assisted carbohydrate electrophoresis (FACE) to directly measure the predominate disaccharide of unfractionated heparin. In this study, unfractionated heparin was measured in vitro throughout a wide range of heparin concentrations in plasma. Seven in vivo pharmacokinetic studies in five normal subjects given 3,000 USP units of unfractionated heparin intravenously showed a three-phase elimination process with higher peak plasma levels and shorter elimination times than predicted from previous studies. At these doses, heparin is largely eliminated intact through urinary excretion. Body weight has a significant effect on heparin kinetics. When we compared the direct biochemical assay with two biological clotting assays, we found the latter can overestimate biochemical heparin concentrations. The FACE assay, due to its sensitivity, is also able to measure circulating levels of endogenous heparin in plasma and urine. Direct heparin measurement using the FACE technique is practical and useful for studies of the correlation of biochemical and biological activities.

Elucidation of N-linked oligosaccharide structures of recombinant human factor VIII using fluorophore-assisted carbohydrate electrophoresis.

Characterization of the carbohydrate moiety is a critical measure of manufacturing process consistency of recombinant human Factor VIII (rFVIII) in Chinese-hamster ovary (CHO) cells. FVIII, a large (300 kDa) glycoprotein, is employed therapeutically for the correction of haemophilia A. While N-linked and O-linked oligosaccharides are found in this protein, the current study focuses on the N-linked oligosaccharides. The N-linked oligosaccharides from rFVIII were released using either peptide N-glycosidase F or endoglycosidase H, derivatized with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonate, and analysed by fluorophore-assisted carbohydrate electrophoresis (FACE). The electrophoretically resolved oligosaccharide bands were isolated and individual bands subjected to digestion with defined pools of exoglycosidases and re-electrophoresed on FACE sequencing gels. The resulting gel patterns were interpreted, based on band mobility shifts, to obtain the sequence structure of the oligosaccharides. A total of eight acidic and 12 neutral structures were identified, and the majority of the oligosaccharides (approximately 92%) were found to be sialylated. All of the major oligosaccharide structures found in CHO-cell-derived rFVIII have also been reported to be present in plasma-derived FVIII. Among the most abundant are disialylated, biantennary, core-fucosylated (approximately 40%), followed by trisialylated, triantennary, core-fucosylated and monosialo, biantennary, core-fucosylated structures (each approximately 18%). The Gal alpha 1-3Gal structures reported to be present in baby-hamster-kidney-cell-derived rFVIII were not found in the CHO-cell-derived protein. The glycosylation patterns were consistent in six random lots of rFVIII [coefficient of variation (%) 3-14] based on percentage lane luminance data of bands that represent approximately 98% of all asparagine-linked oligosaccharides.

Mice lacking both subunits of lysosomal beta-hexosaminidase display gangliosidosis and mucopolysaccharidosis.

The GM2 gangliosidoses, Tay-Sachs and Sandhoff diseases, are caused by mutations in the HEXA (alpha-subunit) and HEXB (beta-subunit) genes, respectively. Each gene encodes a subunit for the heterodimeric lysosomal enzyme, beta-hexosaminidase A (alpha beta), as well as for the homodimers beta-hexosaminidase B (beta beta) and S (alpha alpha). In this study, we have produced mice that have both Hexa and Hexb genes disrupted through interbreeding Tay-Sachs (Hexa-/-) and Sandhoff (Hexb-/-) disease model mice. Lacking both the alpha and beta-subunits these 'double knockout' mice displayed a total deficiency of all forms of lysosomal beta-hexosaminidase including the small amount of beta-hexosaminidase S present in the Sandhoff disease model mice. More surprisingly, these mice showed the phenotypic, pathologic and biochemical features of the mucopolysaccharidoses, lysosomal storage diseases caused by the accumulation of glycosaminoglycans. The mucopolysaccharidosis phenotype is not seen in the Tay-Sachs or Sandhoff disease model mice or in the corresponding human patients. This result demonstrates that glycosaminoglycans are crucial substrates for beta-hexosaminidase and that their lack of storage in Tay-Sachs and Sandhoff diseases is due to functional redundancy in the beta-hexosaminidase enzyme system.

Fluorophore-assisted carbohydrate electrophoresis in the separation, analysis, and sequencing of carbohydrates.

Carbohydrate analysis has traditionally been viewed as a specialty science, performed only in a few well-established laboratories using conventional carbohydrate analysis technology (e.g. NMR, gas chromatography-mass spectroscopy, high-performance liquid chromatography, capillary electrophoresis) combined with the specialized technical training that has been essential for accurate interpretation of the data. This tradition of specialized laboratories is changing, due primarily to an increase in the number of scientists performing routine carbohydrate analysis. As a result, many scientists who are not trained in traditional carbohydrate analytical techniques now need to be able to perform accurate carbohydrate analysis in their own laboratories. This has created a need for technically simple and inexpensive methods of carbohydrate analysis. In this review, we present application vignettes of a technically simple, yet analytically powerful method called fluorophore-assisted carbohydrate electrophoresis (FACE). FACE can be used for performing routine oligosaccharide profiling, monosaccharide analysis, and sequencing of a variety of carbohydrates.

Characterization of nuclear pore protein p62 produced using baculovirus.

Nuclear pore glycoproteins are essential components of the nuclear import apparatus in eukaryotes. In vertebrates, the most abundant of these glycoproteins is a molecule called p62. Like other O-linked N-acetylglucosamine glycoproteins, p62 is normally modified in the cytoplasm and cannot be overexpressed and conveniently collected in a secreted form. We devised an efficient scheme for expression and purification of recombinant p62 from Sf9 cells that may have general applicability for this class of glycoproteins. The purified rat p62 bound to wheat germ agglutinin, consistent with modification by O-linked N-acetylglucosamine. Carbohydrate analysis, in conjunction with amino acid analysis, revealed that baculovirus-expressed rat p62 contains 5-6 mol of N-acetylglucosamine/mol of p62. As observed by circular dichroism, purified p62 expressed in the baculovirus system or in Escherichia coli share essentially the same secondary structure. Purified glycosylated rat p62 will be critical in determining the role of N-acetylglucosamine in both nuclear transport and assembly of the nuclear pore complex.

Analysis of nuclear pore protein p62 glycosylation.

Glycoprotein components of the nuclear pore are essential for nuclear transport and are modified by both glycosylation and phosphorylation. The function and control of these post-translational modifications are poorly understood. Glycosylation of the major rat nuclear pore glycoprotein, p62, was examined in vitro using recombinant p62 as a substrate. Rat p62 was expressed in Escherichia coli and purified to near homogeneity. Kinetic analysis using a partially purified mammalian transferase suggests that the recombinant protein is an excellent substrate (Km = 0.30 microM) for the transfer of GlcNAc from UDP-GlcNAc (Km = 1.8 microM). Localization of the sites of O-linked GlcNAc glycosylation of rat p62 was performed by a combination of deletion analysis of in vitro translation products and by immunoprecipitation of [14C]GlcNAc-labeled proteolytic fragments. The amino terminus of rat p62 is poorly glycosylated with no O-linked GlcNAc sites between Lys22 and Lys97; the carboxyl terminus has one known glycosylation site at Ser471. The majority of the glycosylation sites in rat p62 are likely to occur on the six clustered Ser residues in the central Ser/Thr-rich region from Ser270 to Thr294. A synthetic peptide derived from this region is a good substrate for O-GlcNAc addition (Km = 30 microM) and a potent competitive inhibitor of p62 glycosylation (Ki = 15 microM). It is proposed that this Ser/Thr-rich domain functions as a linker region between the amino-terminal beta-pleated sheet and the carboxyl terminal alpha-helical domains. O-Glycosylation and phosphorylation of this linker region could provide a dynamic means of altering the conformation of p62 during nuclear pore assembly and disassembly.

Capillary electrophoresis separation of oligosaccharides: I. Effect of operational variables.

The influence of operating variables on the separation of labeled oligosaccharide molecules in capillary electrophoresis with polymer networks is presented. In this study, an equation was derived that relates the effects of electrophoresis variables such as field strength, temperature, molecular weight and gel concentration to the migration velocity. As a model system, 8-aminonaphthalene-1,3,6-trisulfonate ANTS-labeled wheat starch digest was examined to show the validity of the equation. As an illustration of the high resolving power achievable with capillary electrophoresis, oligosaccharide molecules up to the degree of polymerization of 25 were separated with the very high efficiency of N > 10(6) and efficiency generation rate of 3500 plates/m/s for maltose. Separation based on the size of the oligosaccharides was not obtained with either entangled or unentangled polymer solutions.

The gene encoding rat nuclear pore glycoprotein p62 is intronless.

Glycoproteins of the nuclear pore complex are thought to play an important role in the transport of regulatory proteins and ribonucleoproteins across the nuclear envelope. However, the genetic elements and signals that control the expression of nuclear pore glycoproteins are poorly understood. To study the transcriptional regulation of mammalian nuclear pore glycoprotein biosynthesis, we have isolated the gene coding for the major rat nuclear pore glycoprotein p62. The p62 gene consists of a 2941-base pair region that is linear with the full length p62 cDNA with no intervening sequences. Quantitative Southern analysis revealed that the gene is present in single copy. The p62 gene encodes a 525-amino acid open reading frame that directs the synthesis of the 62-kDa pore glycoprotein in vitro and in transfected cultured cells. The 5'-flanking region contains two potential transcription start sites; primer extension analysis revealed that the furthest upstream site is preferentially used in vivo. When linked to a reporter gene, the 5'-flanking region of the p62 gene serves as an active promoter.

Structure and function of the nuclear pore complex: new perspectives.

The double membrane of the nuclear envelope is a formidable barrier separating the nucleus and cytoplasm of eukaryotic cells. However, movement of specific macromolecules across the nuclear envelope is critical for embryonic development, cell growth and differentiation. Transfer of molecules between the nucleus and cytoplasm occurs through the aqueous channel formed by the nuclear pore complex (NPC). Although small molecules may simply diffuse across the NPC, transport of large proteins and RNA requires specific transport signals and is energy dependent. A family of pore glycoproteins modified by O-linked N-acetylglucosamine moieties are essential for transport through the NPC. Recent evidence suggests that the regulation of nuclear transport may also involve the interaction of RNA and nuclear proteins with specific binding proteins that recognize these transport signals. Are these nuclear pore glycoproteins and signal binding proteins the 'gatekeepers' that control access to the genetic material? Recent evidence obtained from a combination of biochemical and genetic approaches suggests--perhaps.

Primary sequence and heterologous expression of nuclear pore glycoprotein p62.

The major nuclear pore protein p62 is modified by O-linked N-acetylglucosamine and functions in nuclear transport. We have cloned, sequenced, and expressed the full-length rat p62 cDNA. The rat p62 mRNA is 2,941 nucleotides long and encodes a protein of 525 amino acids containing 30% serine and threonine residues. The amino acid sequence near the amino-terminus contains unique tetrapeptide repeats while the carboxy-terminus consists of a series of predicted alpha-helical regions with hydrophobic heptad repeats. Heterologous expression of rat p62 in African Green Monkey Kidney COS-1 cells and CV-1 cells was detected using a species-specific antipeptide serum. When transiently expressed in COS-1 cells, rat p62 binds wheat germ agglutinin and concentrates at the spindle poles during mitosis. In CV-1 cells cotransfected with rat p62 cDNA and SV40 viral DNA, rat p62 associates with the nuclear membrane without interfering with the nuclear transport of SV40 large T antigen. The ability to express p62 in tissue culture cells will facilitate analysis of the role of this pore protein in nuclear transport.

Glycosylation of nuclear pore protein p62. Reticulocyte lysate catalyzes O-linked N-acetylglucosamine addition in vitro.

The addition of O-linked N-acetylglucosamine (GlcNAc) to the major nuclear pore complex glycoprotein p62 was examined. Expression of the rat p62 cDNA in transfected monkey cells was detected using a rat p62-specific antipeptide antiserum and two previously described nuclear pore-specific monoclonal antibodies which require O-linked GlcNAc for binding. Although the p62 cDNA was predicted to encode a 54-kDa polypeptide, the product expressed in monkey cells migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two species of 62 and 59-kDa. Cell-free translation of the p62 in vitro transcript yielded a 59-kDa polypeptide using wheat germ extract and a 62-kDa product using a commercially available rabbit reticulocyte lysate. Several lines of evidence indicated that the 62-kDa rabbit reticulocyte lysate translation product was modified by O-linked N-acetylglucosamine; the protein bound specifically to a wheat germ agglutinin affinity column and was converted to 59 kDa when treated with jack bean beta-acetylglucosaminidase. The 59-kDa unglycosylated wheat germ translation product was converted to the 62-kDa glycosylated form upon incubation with reticulocyte lysate demonstrating that O-linked GlcNAc can be added to p62 post-translationally.

Analysis of actin and tropomyosin in hearts of cardiac mutant axolotls by two-dimensional gel electrophoresis, western blots, and immunofluorescent microscopy.

When homozygous, recessive mutant gene c in Ambystoma mexicanum results in a failure of embryonic heart function. This failure is apparently due to abnormal inductive influences from the anterior endoderm resulting in an absence of normal sarcomeric myofibril formation. Biochemical and immunofluorescent studies were undertaken to evaluate the contractile proteins actin and tropomyosin in normal and mutant hearts. For the immunofluorescent studies, cardiac tissues were fixed in periodate-lysine-paraformaldehyde, frozen sectioned, and immunostained by an indirect method with monospecific polyclonal antibodies produced against highly purified chicken heart actin and tropomyosin. In normal hearts, both antiactin and antitropomyosin stained the myofibrillar I-bands intensely. In mutant hearts, intensity of staining with antiactin antibody was similar to normal, although sarcomeric patterns were not observed. Staining intensity for tropomyosin with antitropomyosin antibody was significantly reduced in mutant hearts when compared to normal. Biochemical studies were used to evaluate antibody specificity, antigenic variability, and relative protein concentrations of actin and tropomyosin in normal and mutant cardiac tissues. Tissue homogenates were electrophoresed in two dimensions, and second-dimension slab gels were either Coomassie Blue silver-stained or transblotted onto nitrocellulose and the proteins stained with antibodies. Stained gels and immunoblots of cardiac proteins reveal that the amounts of actin isoforms are identical in normal and mutant hearts. However, these methods demonstrate a significantly reduced amount of tropomyosin in mutant tissue. This confirms earlier studies suggesting reduced amounts of tropomyosin in mutant hearts based upon immunological assays. Thus, failure of normal myofibrillogenesis in gene c mutant hearts does not appear to result from a change in actin isoform composition but may be related to a deficiency in tropomyosin.

Partial cDNA sequence encoding a nuclear pore protein modified by O-linked N-acetylglucosamine.

The nuclear pore complex contains a family of proteins ranging in molecular mass from 35 to 220 kDa that are glycosylated with O-linked N-acetylglucosamine (GlcNAc) residues. We sought to determine the primary sequence of a nuclear pore protein modified by O-linked GlcNAc. The major (62 kDa) nuclear pore glycoprotein (np62) was purified from rat liver nuclear envelopes by immunoaffinity chromatography and preparative gel electrophoresis. After CNBr fragmentation, a glycopeptide was isolated and microsequenced. An oligonucleotide probe based on this sequence information was used to screen a lambda gt11 cDNA library constructed from poly(A) mRNA of the rat thyroid cell line FRTL-5. A clone (B5) was isolated and shown to hybridize to a single 2.5-kilobase species in poly(A) mRNA from rat liver and FRTL-5. This insert was sequenced and found to contain a 691-base-pair cDNA encoding a 155-amino acid open reading frame. This open reading frame contained a CNBr fragment identical to the original glycopeptide sequence and a second CNBr fragment corresponding to a nonglycosylated peptide that was also isolated from the purified pore glycoprotein. The B5 cDNA produced a beta-galactosidase fusion protein of the size predicted by the open reading frame. Analysis of the residues making up a presumptive glycosylation site suggests that the sequence is unlike any known sites for enzymatic N- or O-linked glycosylation. The partial sequence of the 62-kDa nuclear pore glycoprotein shows little similarity to other characterized proteins and elucidates structural features of a member of the family of nuclear pore glycoproteins.

Regulation of dolichyl phosphate-mediated protein glycosylation: estrogen effects on glucosyl transfers in oviduct membranes.

Regulation of Glc transfer from UDP-Glc via Glc-P-Dolichol to form Glc3-Man9-oligosaccharide-lipid has been studied during estrogen-induced chick oviduct differentiation. The process was studied as two distinct reactions: transfer of Glc from UDP-Glc to Dol-P, forming Glc-P-Dol; and transfer of Glc from Glc-P-Dol to Man9-OL (oligosaccharide-lipid), forming a series of glucosylated oligosaccharide-lipids. Kinetic analysis of [14C]Glc transfer from UDP-[14C]Glc to endogenous Dol-P shows that Dol-P is limiting in membrane preparations and that, concomitant with estrogen-induced differentiation, there is an increase in Dol-P available for Glc transfers. There is also greater glucosyl transferase activity present in membranes from mature hens and estrogenized chicks than in membranes from immature chicks. In order to study the second phase of glucosylation, transfer to the oligosaccharide, it was necessary to develop an assay in which membranes could be reacted with exogenously added substrates, [14C]Glc-P-Dol and [3H]Man9-OL. This reaction is dependent on detergent (0.02% NP-40 was used) and is stimulated by EDTA. The apparent Km for Glc-P-Dol was about 1.5 microM. A series of double-labeled oligosaccharides having sizes consistent with Glc1-, Glc2-, and Glc3-Man9-OL were formed. Chemical and enzymatic analysis of [14C]Glc oligosaccharides formed by incubation with the exogenous substrates, or by incubation with UDP-[14C]Glc and endogenous acceptors, indicated that the glucosylated oligosaccharides were similar. Assays of membranes from estrogenized chicks, mature hens, and hormone-withdrawn chicks showed increased glucosyl transferase activity upon hormone treatment. Similar assays in the absence of exogenous Man9-OL indicated that hormone treatment was also accompanied by increased levels of endogenous oligosaccharide-lipid acceptors.

Effects of dietary fiber on intestinal ion fluxes in rats.

The effect of short-term fiber ingestion on jejunal ion fluxes was evaluated in rats using a standard Ussing chamber technique. Ingestion of cellulose and pectin decreased the mucosal to serosal fluxes of both Na and Cl but did not significantly alter serosal to mucosal fluxes; net fluxes of both Na and Cl were significantly lower in the group supplemented with dietary fiber as compared to those animals fed a fiber-free diet. Both potential difference and short-circuit current were higher in the fiber-free group than in the group supplemented with dietary fiber; tissue conductance, however, was unaffected by fiber ingestion. The residual flux of all three groups was virtually identical suggesting that electrical alterations observed after cellulose and pectin ingestion are not the result of ion fluxes other than Na and Cl. These data, coupled with previous observations that short-term fiber supplementation impairs glucose and leucine absorption, suggest that fiber ingestion alters the intestinal membrane, specifically sites of active transport.

Phosphatidylcholine synthesis in the developing small intestine.

1. Phosphatidylcholine synthesis in the foetal, newborn and adult small intestine of rats was studied by determination of cytidine diphosphocholine-1,2-diacylglycerocholine phosphotransferase (cholinephosphotransferase) and acyl-CoA-1-acyl-sn-glycerol-3-phosphocholine acyltransferase (lysophosphatidylcholine acyltransferase) activities and the incorporation of [1-14C]oleic acid into phosphatidylcholine. 2. Cholinephosphotransferase activity was low in foetal jejunum and ileum, increased 3-4 fold in the ileum by 6 days of age and by 12 days in the jejunum. Jejunal activity remained constant throughout weaning; ileal activity gradually decreased to values 25% of that of the jejunum. 3. Lysophosphatidylcholine acyltransferase activity was high in foetal jejunum and ileum, decreased 70% immediately after birth in the jejunum and increased to adult values by 12 days of age. Ileal activity decreased by 20% after birth, but decreased more rapidly at weaning to 30% of the activity in jejunum. 4. Initial rates and steady-state incorporation of [1-14C]oleic acid into phosphatidylcholine by jejunal rings of 10 day-old rats exceeded that observed in jejunal rings from adult rats by 2-4-fold. 5. In the postnatal jejunum, neither cholinephosphotransferase and lysophosphatidylcholine acyltransferase activities nor oleic acid incorporation were stimulated by cortisone administration in vivo.