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Recently, our group reported the expression of recombinant human erythropoietin in goat milk (rhEPO-milk) as well as in the mammary epithelial cell line GMGE (EPO-GMGE) by cell culture using the adenoviral transduction system. N-Glycosylation characterization of rhEPO-milk by Normal-Phase HPLC profiling of the fluorophore, 4-aminobenzoic acid-labeled enzymatically released N-glycan pool from rhEPO-goat milk, combined with MALDI, ESI-MS and LC/MS, revealed that low branched, core-fucosylated, N-glycans predominate. The labeled N-glycans were separated into neutral and charged fractions by anion exchange chromatography and the charged N-glycans were found to be mostly alpha2,6-monosialylated with Neu5Ac or Neu5Gc in a ratio of 1:1. Unlike the N-glycans from rhEPO produced in CHO cells, where the glycans are multiantennary highly sialylated, core-fucosylated oligosaccahrides, or even in the goat mammary gland epithelial cell line cultured in vitro in which multiantennary, core- and outer-arm fucosylated, monosialylated N-glycans are the most abundant species, a large proportion of the N-glycans from rhEPO-milk were monosialylated, biantennary, antennae mostly terminating with the more unusual GalNAc-GlcNAc motive and without outer-arm fucosylation. These findings, emphasizing the difference in the N-glycan repertoire between the rhEPO-milk and EPO-GMGE, are consistent with the principle that glycosylation is cell-type dependent and that the cell environment is crucial as well.

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We have established a continuous, non-transformed cell line from primary cultures from Capra hircus mammary gland. Low-density cultures showed a homogeneous epithelial morphology without detectable fibroblastic or myoepithelial cells. The culture was responsive to contact inhibition of proliferation and its doubling time was dependent on the presence of insulin and epidermal growth factor (EGF). GMGE cells secrete caseins regardless of the presence or absence of lactogenic hormones in the culture media. Investigation of the total N-glycan pool of human erythropoietin (rhEPO) expressed in GMGE cells by monosaccharide analysis, HPLC profiling, and mass spectrometry, indicated significant differences with respect to the same protein expressed in Chinese hamster ovary (CHO) cells. N-Glycans of rhEPO-GMGE are core-fucosylated, but fucosylation of outer arms was also found. Our results also revealed the presence of low levels of sialylation (>95% Neu5Ac), N,N'-diacetyllactosediamine units, and possibly Gal-Gal non-reducing terminal elements.

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Coeliac disease (CD) is described as an autoimmune enteropathy associated with the presence of IgG and IgA antigliadin and antitransglutaminase autoantibodies. While of diagnostic significance, the role of these autoantibodies in the immunopathogenesis of CD is elucidated. An inappropriate T cell immune response to gluten is also involved in the pathogenesis of CD, as evidenced by autoantibody switching. The N-glycans released from serum IgG of CD patients and three groups of healthy controls, of differing age ranges, were analysed by NH2-high performance liquid chromatography (HPLC). The fucosylated biantennary N- glycans were the most abundant neutral oligosaccharides; in particular, the agalacto form (G0F) showed a mean value of 42% (s.d. +/- 7.4), 30% (s.d. +/- 5.9), 26% (s.d. +/- 4.2) and 35% (s.d. +/- 6.8) for CD patients, healthy children, healthy adults under 40 and healthy adults over 40 years old, respectively. The ratio of asialo agalacto fucosylated biantenna to asialo monogalacto fucosylated biantenna (G0F)/(G1F) for CD patients showed a significant increase compared to healthy children (P < 0.0002), healthy adults under 40 (P < 0.0002) and healthy adults over 40 years old (P < 0.01). Hypogalactosylation was more pronounced for CD patients than for the patients with other autoimmune diseases such as rheumatoid arthritis or psoriatic arthritis.

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Using anion-exchange chromatography the catalyticdomain of endoglucanase 1 (Cel7B) from Trichoderma reesei was resolved in multiple fractions with different isoelectric points, presumably related to different glycoforms of the enzyme. The protein fractions were analysed using lectins and electrospray MS. Isolated N-glycans were analysed by fluorophore-assisted carbohydrate electrophoresis and amine-adsorption HPLC. The results show that this particular preparation contained at least 14 different glycoforms. The major isoform contained only one GlcNAc, presumably N-linked, and one mannose, most probably O-linked to serine/threonine at a separate site. Except for a small population containing Man(5)GlcNAc(2)+1-2 Man, the rest of the protein had negatively charged phosphate-containing N-glycans. All glycoforms contained at least one O-linked mannose residue. The increased negative charge of the protein, introduced by oligosaccharide phosphorylation, is the most probable reason for the different isoelectric points and the occurrence of multiple peaks during purification.

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Differences in glycosylation between the natural alpha-1,6 glucan-6-glucanohydrolase from Penicillium minioluteum and the heterologous protein expressed in the yeast Pichia pastoris were analyzed. Glycosylation profiling was carried out using fluorophore-assisted carbohydrate electrophoresis and amine absorption high-performance liquid chromatography (NH(2)-HPLC) in combination with matrix-assisted laser desorption-time of flight-mass spectrometry. Both microorganisms produce only oligomannosidic type structures, but the oligosaccharide population differs in both enzymes. The native enzyme has mainly short oligosaccharide chains ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2), of which Man(8)GlcNAc(2) was the most represented oligosaccharide. The oligosaccharides linked to the protein produced in P. pastoris range from Man(7)GlcNAc(2) up to Man(14)GlcNAc(2), with Man(8)GlcNAc(2) and Man(9)GlcNAc(2) being the most abundant structures. In both enzymes the first glycosylation site (Asn(5)) is always glycosylated. However, Asn(537) and Asn(540) are only partially glycosylated in an alternate manner.

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The aspartic proteinase gene of Mucor pusillus rennin expressed in Pichia pastoris was characterized in terms of structural and conformational stability induced by temperature. This enzyme is 12% glycosylated, with a similar specific activity to the native fungal enzyme. The secondary structure determined by CD is mainly due to beta-sheet structures with an important contribution of aromatic components. The calorimetric studies were carried out in the temperature range in which the enzyme is most stable. The enzyme undergoes an irreversible, highly scan-rate-dependent thermal denaturation under all the experimental conditions investigated. Between pH 3.0 and 7.0, only one endotherm characterized the thermal denaturation of enzyme. At pH 5.0, the most stable condition found, the denaturation can be fitted to the two-state irreversible model. Thus the kinetic constant and activation parameters of the denaturation process could be obtained. Upon reaching pH 7.5, the denaturation is characterized by two endotherms. This evidence indicates the complex tridimensional structure of this enzyme. Finally, taking into account the conservative tertiary structure of the aspartic proteinase family we comment on our results with reference to the crystallographic structure of M. pusillus pepsin [Newman, Watson, Roychowdhury, Badasso, Cleasby, Wood, Tickle and Blundell (1993) J. Mol. Biol. 221, 1295-1309].

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Aspartic protease, widely used as a milk-coagulating agent in industrial cheese production, contains three potential N-glycosylation sites. In this study, we report the characterization of N-linked oligosaccharides on recombinant aspartic protease secreted from the methylotrophic yeast Pichia pastoris using a combination of mass spectrometric, 2D chromatographic, chemical and enzymatic methods. The carbohydrates from site I (Asn79) were found to range from Man6-17GlcNAc2 with 50% bearing a phospho-diester-motif, site II (Asn113) was not occupied and site III (Asn188) contained mostly uncharged species ranging from Man-13GlcNAc2. These charged groups are not affecting the transport through the secretion pathway of the recombinant glycoprotein. Changes from a molasses-based medium to a minimal salts-based medium led to a clear reduction of the degree of phosphorylation of the N-glycan population.

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We report the characterization of N-linked oligosaccharides on six foreign glycoproteins secreted from the methylotrophic yeast Pichia pastoris. These proteins included: a bacterial enzyme, Bacillus licheniformis alpha-amylase; three fungal enzymes, Saccharomyces cerevisiae invertase, Penicillium minioluteum dextranase, and Mucor pusillus aspartic protease; and two higher eukaryotic proteins, Boophilus microplus (tick) gut antigen and bovine enterokinase catalytic subunit. The carbohydrates on these proteins were observed to vary in size, with Man8GlcNAc2 and Man9GlcNAc2 structures being the most frequently observed species. Substantial amounts of shorter oligomannoside structures were present only on invertase, and longer structures (up to Man18GlcNAc2) were common on aspartic protease and enterokinase. Phosphorylated oligosaccharides were observed on one protein, aspartic protease. Unlike oligosaccharides on glycoproteins secreted from S. cerevisiae, no terminal alpha1,3-linked mannosylation was observed on any of the six P. pastoris-secreted proteins. Changing the growth and induction medium from a minimal salt-based medium to a molasses-based medium had little effect on the size of the oligomannosides. From these results, it is apparent that most foreign proteins secreted from P. pastoris are not subjected to the extensive mannosylation (hyperglycosylation) that commonly occurs in proteins secreted from S. cerevisiae.

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We describe a simple and sensitive two-dimensional sugar-mapping technique of 8-amine-1,3,6-naphthalene trisulfonic acid derivatives (ANTS derivatives) of neutral and sialyloligosaccharides for structure analysis and characterization of N-linked oligosaccharides using picomoles of samples. The method includes: (1) reductive amination with ANTS of enzymatically released oligosaccharides, (2) simultaneous separation of oligosaccharide derivatives in a fluorophore-assisted carbohydrate electrophoresis and NH2-HPLC column under ion suppression conditions, (3) plotting of the relative migration indexes (X axis) and relative retention times (Y axis), and (4) when necessary, additional exoglycosidase digestion. As illustrated by the glycosylation profiling and structural analysis of alpha 1 anti-trypsin and murine IgG 2a, this methodology fulfills most of the requirements for a complete characterization of neutral and charged oligosaccharides released from N-glycosylated glycoprotein.

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A periplasmic invertase from the yeast Candida utilis was purified to homogeneity from cells fully derepressed for invertase synthesis. The enzyme was purified by successive Sephacryl S-300, and affinity chromatography and shown to be a dimeric glycoprotein composed of two identical monomer subunits with an apparent molecular mass of 150 kDa. After EndoH treatment, the deglycosylated protein showed an apparent molecular weight of 60 kDa. The apparent K(m) values for sucrose and raffinose were 11 and 150 mM, respectively, similar to those reported in Saccharomyces cerevisiae. The range of optimum temperature was 60-75 degrees C. The optimum pH was 5.5 and the enzyme was stable over pH range 3-6.

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The DEX gene encoding an extracellular dextranase was isolated from the genomic DNA library of Penicillium minioluteum by hybridization using the dextranase cDNA as a probe. Comparison of the gene and cDNA sequences revealed that the DEX gene does not contain introns. Amino acid sequences comparison of P. minioluteum dextranase with other reported dextranases reveals a significant homology (29% identity) with a dextranase from Arthrobacter sp. CB-8. The DEX gene fragment encoding a mature protein of 574 amino acids was expressed in the methylotrophic yeast Pichia pastoris by using the SUC2 gene signal sequence from Saccharomyces cerevisiae under control of the alcohol oxidase-1 (AOX1) promoter. Over 3.2 g/l of enzymatically active dextranase was secreted into the medium after induction by methanol. The yeast product was indistinguishable from the native enzyme in specific activity and the N-terminus of both proteins were identical.

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A DNA fragment containing a transcription regulating region of the alcohol oxidase (AOX1) gene from the methylotrophic yeast Pichia pastoris was used in the construction of a vector for the expression of heterologous proteins in the methylotrophic yeast Hansenula polymorpha. We used this vector to clone the SUC2 gene from Saccharomyces cerevisiae into H. polymorpha yeast. The culture conditions for invertase production using a fed-batch culture were studied. More than 1.5 x 10(3) U/ml of biologically active invertase (1 g/l) were secreted to the cellular periplasmic space. The fermentative process was scaled up to 50 l. Invertase produced from H. polymorpha was glycosylated, but it contained significantly less carbohydrate than protein produced by S. cerevisiae. Using the Western-blot technique, it was observed that invertase secreted from H. polymorpha and invertase secreted from S. cerevisiae showed common antigenic determinants.

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In the present paper we report the biochemical characteristics of the recombinant tick (Boophilus microplus) gut antigen Bm86 that previously has been cloned, expressed and recovered at high levels in the methylotrophic yeast Pichia pastoris. The results demonstrate that rBm86 had a modification at position 92 (Thr replaced by Ile) and aggregated, forming particles ranging between 17 and 40 nm. The rBm86 was N-glycosylated, having at least two non-glycosylated sequons (Asn-329 and Asn-363) and a ratio of only 0.4/65 (free Cys/total Cys)/mol of protein.

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We report here a methodology that allows the identification of glycosylation sites by a combination of protein enzymatic digestion, glycopeptide separation on a reverse-phase HPLC column, and further recognition in a dot-blot system using concanavalin A-horseradish peroxidase. Wheat germ agglutinin-horseradish peroxidase is used as the recognition system for peptides generated after proteolytic digestion of endoglycosidase H deglycosylated protein. Glycosylation sites were confirmed by automatic Edman degradation and fast atom bombardment mass spectrometry. This methodology was applied to a model glycoprotein, alpha-amylase from Bacillus licheniformis, which is unglycosylated in its natural host and appears highly glycosylated when expressed in the methylotrophic yeast Pichia pastoris.

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The cDNA of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium has been cloned and sequenced. The 5' end was obtained by PCR amplification. The cDNA contains 2310 translated bases excluding the poly(A) tail. The deduced mature protein contains 770 amino acid residues and is preceded by a 18 residue long signal peptide. The regions of the amino acid sequence corresponding to the heme and FAD domains of CDH were identified as well as the nucleotide-binding motif, the disulfide pairing and a methionine residue chelating the heme iron. No homologous sequences were found for the heme domain, however, the FAD domain appears to be distantly related to the GMC oxidoreductase family.

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We have cloned and expressed a bacterial thermostable alpha amylase gene in Pichia pastoris using the methanol-controlled alcohol oxidase (AOX1) promoter. Two integrative vectors were constructed with two different secretion signal sequences in order to obtain efficient secretion of the protein. One vector contains the structural gene encoding the mature alpha amylase fused to the SUC2 gene signal sequence from Saccharomyces cerevisiae. In the other vector, the alpha amylase is expressed with its own signal sequence. In both cases, the alpha amylase were secreted into the culture medium with high efficiency, around 2.5 and 0.9 g/l respectively.

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Recently, a gene coding for the Bm86 tick gut glycoprotein was cloned, expressed in Escherichia coli and shown to induce an immunological response in cattle to damage ticks engorging on these animals (Rand et al., 1989). We report here the increased expression of the Bm86 antigen from the cattle tick Boophilus microplus in the methylotrophic yeast Pichia pastoris. The recombinant protein was obtained with a purity higher than 95% by a procedure with a high yield. The conducted biochemical studies demonstrated the antigen to be glycosylated and found to form particles of around 17 to 45 nm in diameter with enhanced immunogenic properties. Ticks engorging on vaccinated cattle were significantly damaged as a result of the immune response against the recombinant antigen. This system permits the obtainment in a high yield of the tick Bm86 antigen, in a glycosylated and particulated form.

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