Galactofuranoic-oligomannose N-linked glycans of alpha-galactosidase A from Aspergillus niger.

Extracellular alpha-galactosidase A was purified from the culture filtrate of an over-producing strain of Aspergillus niger containing multiple copies of the encoding aglA gene under the control of the glucoamylase (glaA) promoter. Endoglycosidase digestion followed by SDS/PAGE, lectin and immunoblotting suggested that glycosylation accounted for approximately 25% of the molecular size of the purified protein. Monosaccharide analysis showed that this was composed of N-acetyl glucosamine, mannose and galactose. Mild acid hydrolysis, mild methanolysis, immunoblotting and exoglycosidase digestion indicated that the majority of the galactosyl component was in the furanoic conformation (beta-D-galactofuranose, Galf). At least 20 different N-linked oligosaccharides were fractionated by high-pH anion-exchange chromatography following release from the polypeptide by peptide-N-glycosidase F. The structures of these were subsequently determined by fast atom bombardment mass spectrometry to be a linear series of Hex(7-26)HexHA(c2). Indicating that oligosaccharides from GlcNA(c2)Man(7), increasing in molecular size up to GlcNA(c2)Man(24) were present. Each of these were additionally substituted with up to three beta-Galf residues. Linkage analysis confirmed the presence of mild acid labile terminal hexofuranose residues. These results show that filamentous fungi are capable of producing a heterogeneous mixture of high molecular-size N-linked glycans substituted with galactofuranoic residues, on a secreted glycoprotein.

The effect of pH on glucoamylase production, glycosylation and chemostat evolution of Aspergillus niger.

The effect of ambient pH on production and glycosylation of glucoamylase (GAM) and on the generation of a morphological mutant produced by Aspergillus niger strain B1 (a transformant containing an additional 20 copies of the homologous GAM glaA gene) was studied. We have shown that a change in the pH from 4 to 5.4 during continuous cultivation of the A. niger B1 strain instigates or accelerates the spontaneous generation of a morphological mutant (LB). This mutant strain produced approx. 50% less extracellular protein and GAM during both chemostat and batch cultivation compared to another strain with parental-type morphology (PS). The intracellular levels of GAM were also lower in the LB strain. In addition, cultivation of the original parent B1 strain in a batch-pulse bioreactor at pH 5.5 resulted in a 9-fold drop in GAM production and a 5-fold drop in extracellular protein compared to that obtained at pH 4. Glycosylation analysis of the glucoamylases purified from shake-flask cultivation showed that both principal forms of GAM secreted by the LB strain possessed enhanced galactosylation (2-fold), compared to those of the PS. Four diagnostic methods (immunostaining, mild methanolysis, mild acid hydrolysis and beta-galactofuranosidase digestion) provided evidence that the majority of this galactose was of the furanoic conformation. The GAMs produced during batch-pulse cultivation at pH 5.5 similarly showed an approx. 2-fold increase in galactofuranosylation compared to pH 4. Interestingly, in both cases the increased galactofuranosylation appears primarily restricted to the O-linked glycan component. Ambient pH therefore regulates both GAM production and influences its glycosylation.

Beta-galactofuranoside glycoconjugates on conidia and conidiophores of Aspergillus niger.

Galactose in the furanoic conformation appears to be limited to bacteria and lower eukaryotes. Galactofuranoic (Galf)-containing glycoconjugates that occur in organisms pathogenic or allergenic to man are frequently antigenic and immunodominant. We have used an immunochemical approach, employing a monoclonal antibody that recognises Galf epitopes, to investigate the presence of Galf-containing glycoconjugates within conidia and conidiophores of Aspergillus niger. ELISA and immunofluorescence microscopy indicated that specific and saturable binding sites were found on both. Inhibition studies confirmed that this binding was to Galf-containing glycoconjugates. Interestingly, the conidiophore heads were particularly rich in these glycoconjugates. Western blotting identified a Galf glycoprotein of 150-200 kDa from disrupted conidia.

An extracellular beta-galactofuranosidase from Aspergillus niger and its use as a tool for glycoconjugate analysis.

Aspergillus niger produces an extracellular beta-galactofuranosidase, which can specifically hydrolyse beta-D-galactofuranose (Galf) from glycoconjugates. The production of this enzyme can be induced by the addition of a Galf-containing A. niger mycelial wall extract. However, on other carbon sources accumulation occurred only during the starvation conditions of the late stationary phase. Extracellular glucoamylases from this stage of cultivation possessed significantly lower levels of Galf than those from the earlier exponential growth phase when beta-galactofuranosidase is absent, suggesting in situ beta-galactofuranosidic hydrolysis. The beta-galactofuranosidase responsible was subsequently purified to homogeneity and characterised. It is a glycoprotein of 90 kDa (determined by SDS-PAGE) with activity against beta-linked Galf residues, with a Km of 4 mM against p-nitrophenyl-beta-D-galactofuranoside and a pH optimum of 3-4. The preparation did not contain other contaminating glycosidase activities; p-nitrophenyl-beta-D- and -alpha-D-galactopyranose, and alpha-D-methyl-Galf were not hydrolysed. Results are presented to show that this enzyme could be employed as a useful tool for the analysis of glycoconjugates containing biologically important Galf components.

An unambiguous microassay of galactofuranose residues in glycoconjugates using mild methanolysis and high pH anion-exchange chromatography.

An original, unambiguous microassay of galactofuranose (Galf) residues in glycoconjugates is described. The method involves mild acid methanolysis (5 mM HCl) for 3 h at 84 degrees C followed by high pH anion-exchange chromatography using a routine monosaccharide system. The methanolysis products Mealpha-Galf and Mebeta-Galf were characterized chromatographically by comparison with the authentic compounds and by their response to treatment with mild acid and with beta-galactofuranosidase. Testing against p-nitrophenyl-beta-Galf and UDPalpha-Galf showed the method to be applicable to both alpha- and beta- galactofuranosides over the range 10-200 pmol. The results of partial mild methanolysis over shorter periods were consistent with initial inversion of anomeric configuration at methylation followed by anomerization to an equilibrium mixture of alpha- and beta-forms. When applied to a sample of invertase from Aspergillus nidulans, the method indicated that all of the mild acid-labile galactose (78% of the total galactose present) was in the form of a galactofuranoside and that much of this was in the beta-configuration. As expected, when applied to asialofetuin (known to contain galactose only in the pyranoside form, Galp), NPalpha-Galp, NPbeta-Galp, or UDPalpha-Galp, mild acid methanolysis failed to produce any galactofuranoside.

Changes in the expression and distribution of fibronectin, laminin and tenascin by cultured fibroblasts from skin lesions of patients with tuberous sclerosis.

Fibronectin, laminin and tenascin play an important part in the assembly of the extracellular matrix and the interaction of cells with it. In this study, changes in their expression and distribution associated with tuberous sclerosis are reported. Fibroblasts from three different tuberous sclerosis skin lesions (forehead plaque, neck fibroma and ungual fibroma) secreted more fibronectin and tenascin into their culture medium than did normal skin fibroblasts. Immunohistochemistry and flow cytometry showed that cells from an ungual fibroma which secreted most of each of these glycoproteins also retained more of them, associated mainly with the cell surface and a perinuclear area. Laminin was also produced by all fibroblasts but only in those from the neck fibroma was more both secreted and retained. The proportions of fibronectin/laminin/tenascin secreted by the skin lesion fibroblasts were markedly different from normal. The results suggest that the characteristic tissue hardening of skin lesions in tuberous sclerosis may result, at least in part, from differences in the expression and distribution of these critical components of the extracellular matrix and its consequent abnormal assembly.

Glucoamylase overexpression and secretion in Aspergillus niger: analysis of glycosylation.

We have studied the effects of overexpression and secretion of a homologous model glycoprotein, glucoamylase (GAM-1), on glycosylation in a single gene copy wild-type parent and multiple gene copy transformants of Aspergillus niger. In batch culture the B36 strain, which possess 80 additional copies of the GAM glaA gene, secreted about 5-8-fold more protein and GAM-1 than the parent strain (N402). A comparison of the glycosylation of GAM-1 secreted by the parent strain with that secreted by the multiple copy and hyper-secreting B36 strain showed that both the N-linked and O-linked glycan composition was very similar. Short oligomannose N-linked glycans were found (Man(7-8)GlcNAc(2)). O-Linked glycans were comprised of short (1-3 residues) oligosaccharide chains of mannose and galactose. Evidence is presented that this galactose is present in the novel galactofuranose conformation. This glycan composition of GAM-1 differed from that of a commercially available (A. niger) GAM source. Microsomes prepared from the mycelium showed a 2-3-fold co-ordinated increase in the activity of the dolichol phosphate:glycosyltransferases. Similar results were obtained from strains B1 (20 copies of glaA) and N402 when grown at a low dilution rate in a chemostat, although both the levels of GAM secretion and the activities of the dolichol phosphate:glycosyltransferases were lower than found in batch culture. These data suggest that A. niger is capable of secreting large amounts of a single glycoprotein combined with higher activity levels of the dolichol phosphate:glycosyltransferases without an increase in the heterogeneity of the glycan structures. Thus, from a biotechnological viewpoint, protein glycosylation may not be a bottleneck to enhanced glycoprotein production using A. niger.

Investigation of the glycosyltransferase enzymes involved in the initial stages of the N-linked protein glycosylation pathway in Aspergillus niger.

A protocol has been developed to produce functional microsomes from mycelium of Aspergillus niger. Using these preparations, radioactive biochemical assays have been designed and optimised to measure the in vitro activities of three of the enzymes of the N-linked dolichol phosphate (Dol-P) glycosylation pathway: UDP-N-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT), Dol-P mannose synthase (DPMS) and Dol-P glucose synthase (DPGS). Exogenous Dol-P and Triton X-100 are essential for in vitro activity. All three Dol-P:glycosyl transferases had alkaline pH optima and activity rapidly decreased below neutrality. Characterisation of the glycolipid products by anion-exchange chromatography and TLC showed that they were Dol-P-P-GlcNAc, Dol-P-Glc and Dol-P-Man for GPT, DPGS and DPMS, respectively. The antibiotic tunicamycin completely inhibited GPT activity at a concentration of 100-200 ng ml(-1) and an IC50 of 40 ng ml(-1), but had little effect on the other two enzymes. The ratio of the activity of the three enzymes to each other suggested that DPMS may be involved in other cellular activities and is probably under different control mechanisms than the other two.

Phorbol ester induces a transient increase in alpha(5)beta(1)-mediated adhesion of the megakaryoblastic cell line CHRF 288-11.

This study investigated the mechanism by which treatment of the CHRF 288-11 megakaryoblastic cell line with phorbol myristate acetate caused a transient increase in adhesion. The adhesion to tissue culture plastic occurred within 4 h and could be reversed by treatment with RGDS-peptide suggesting the involvement of one or more RGD-binding integrins. Ligand-binding adhesion assays suggested that PMA-induced CHRF 288-11 cells had very little affinity for fibrinogen, a low affinity for vitronectin and a much higher affinity for fibronectin. Further adhesion assays performed in the presence of various integrin antagonists or inhibiting monoclonal antibodies demonstrated that the fibronectin-mediated cell adhesion is via the alpha beta , fibronectin receptor, integrin. Flow cytometrical investigations showed that this increase in alpha(5)beta(1)-adhesion on CHRF 288-11 cells following PMA stimulation was not brought about by an increase in alpha(5)beta(1)-integrin expression and inferred that increased adhesion is achieved by an increase in alpha(5)beta(1) ligand-binding function. These findings confirm other reports using different cells that the expression and function of integrins may play an important role in megakaryocytopoiesis. Modulation of integrin function may facilitate the migration of maturing megakaryoblasts in the bone marrow stroma before their movement through the sinus wall and into the blood stream. The report demonstrates that the CHRF 288-11 megakaryoblastic cell line is a useful model for investigating some aspects of these phenomena.

The effect of PMA-induced differentiation on the surface expression of integrins on CHRF 288-11 megakaryoblastic cells in culture.

The integrins GPIIb-IIIa and alpha(v)beta(3) and the integrin subunits alpha(2) , alpha(5) and beta(1) have been shown by flow cytometry to be present on the surface of CHRF 288-11 megakaryoblastic cells grown in culture. PMA-induced differentiation of these cells caused a rapid increase in the surface expression of alpha(v)beta(3), the vitronectin receptor, and a delayed decrease in surface expression of the alpha(5) subunit (presumably present as the alpha(5)beta(1) integrin of the fibronectin receptor). PMA caused no change in expression of GPIIb-IIIa, the alpha(2) subunit (presumably present as alpha(2)beta(1)) or of the beta(1) subunit overall. GPIIb-IIIa appeared to be present as an inactive complex on these cells.

Secretion of two beta-fructofuranosidases by Aspergillus niger growing in sucrose.

Aspergillus niger is induced to secrete two invertases, named SUC 1 and SUC 2, when grown on a minimal medium containing sucrose. Although, both have been classified as beta-D-fructofuranoside fructohydrolases, SUC 2 also possesses inulin hydrolytic activity (sucrose/inulin activity ratio of 4). These activities have been separated from each other and almost completely purified by anion-exchange, lectin affinity chromatography, and chromatofocusing. SUC 1 appeared as a single glycoprotein band on PAGE and SDS-PAGE corresponding in size to 250 and 125 kDa, respectively, compared with a much broader band (suggesting greater glycan heterogeneity) of 210-240 and 90-120 kDa for SUC 2. Therefore, both may be dimers, in their natural conformation. The glycan part of both contained the same monosaccharides: mannose, glucose, galactose, and N-acetylglucosamine; however, SUC 1 had approximately 10-fold more mannose and this was utilized to separate it from SUC 2 by Galanthus nivalis lectin affinity. Both the apparent Km values and the pH activity curves were different; SUC 1 did not show normal Michaelis-Menten kinetics to sucrose and apparent Michaelis constants of 30 and 160 mM were obtained. Activity was observed over a large range of pH 4.5-9 with a maximum at pH 6. In contrast, SUC 2 exhibited a Km of 40 and 1.7 mM to sucrose and inulin, respectively, with a pH optimum of 5.0 for both. Treatment with endo-beta-N-acetylglucosaminidase suggests that in both SUC 1 and SUC 2 some of the glycan present was N-linked glycan but that the differences in enzyme activities were not due to the N-linked moiety.

Changes in the secretion and glycosylation of fibronectin by human skin fibroblasts associated with tuberous sclerosis.

Fibroblasts from skin and skin lesions of patients with tuberous sclerosis (TS) and from skin of normal individuals were grown in culture. ELISA showed that the spent medium of those derived from TS skin lesions contained significantly more fibronectin (FN) than spent medium from the other cells. Amino acid compositional analysis of the FN from TS and normal sources revealed no substantial differences. However the FN of fibroblasts from TS-skin lesions was shown by HPAEC to contain a two- to three-fold increased content of carbohydrate. The changed monosaccharide composition was consistent with an increased content of N- and O-linked glycans and with the former containing polylactosamine chains. Fibroblasts from a normal individual were shown to proliferate more slowly and to produce larger cells when grown on FN from a TS skin lesion compared to growth on FN from normal skin.

Purification and partial characterization of the high and low molecular weight form (S- and F-form) of invertase secreted by Aspergillus nidulans.

Two forms of secreted invertase have been purified from Aspergillus nidulans by ion-exchange and gel-filtration chromatography. S-invertase gave a single, broad, glycoprotein band on PAGE and SDS-PAGE corresponding in size to 185 and 78 kDa, respectively, compared with 94 and 110 kDa for F-invertase. The carbohydrate of S-invertase contained mainly mannose (14%) and less galactose (5%) whereas the F-form yielded mainly galactose (29%) and less mannose (12%). Three sharp bands of enzymically active glycoprotein for both the S-form (pI 4.9-5.2) and the F-form (pI 3-4.2) were observed after isoelectric focusing. Deglycosylation with Endo H simplified this pattern to one enzymically active protein band (pI 5.2). The aglycoenzymes gave narrow bands on PAGE and SDS-PAGE corresponding to 115 kDa and 60 kDa respectively for both S- and F-forms. The specific activity of S-invertase was three-fold higher than that of F-invertase both before and after deglycosylation. The Km values of the two forms of invertase were very similar. Significant homology existed between the N-terminal amino-acid sequences of S-invertase (and of internal peptides derived from it) and sequences of invertase from other species. It is suggested that the higher carbohydrate content in F-invertase results in the native enzyme existing as a monomer and having a greater negative charge and lower specific enzyme activity compared with the dimeric S-enzyme.

Comparison of the N-glycoloylneuraminic and N-acetylneuraminic acid content of platelets and their precursors using high performance anion exchange chromatography.

N-Acetylneuraminic acid (Neu5Ac) and N-glycoloylneuraminic acid (Neu5Gc) are distributed widely in nature. Using a Carbopac PA-1 anion exchange column, we have determined the ratios of Neu5Ac and Neu5Gc in hydrolysates of platelets and their precursors: a rat promegakaryoblastic (RPM) cell line and a human megakaryoblastic leukemia cell line (MEG-01). The ratio of Neu5Gc:Neu5Ac in cultured RPM cells is 16:1, whereas in platelet rich plasma and cultured MEG-01 cells it is 1:38 and 1:28, respectively. The nature of these sialic acids from RPM cells was verified using thin layer chromatography and liquid secondary ion mass spectrometry. The relevance of increased Neu5Gc levels in early stages of development is discussed.

Dolichol: a curriculum cognitionis.

Dolichols were first described about 30 years ago when animal tissues were being examined for the presence of a putative precursor to the polyisoprenoid side chain of ubiquinone. These long-chain 2,3-dihydro-polycis-isoprenoid alcohols are found in all eukaryotic organisms. In many plant tissues they are accompanied by families of other polyisoprenoid alcohols that are usually similar molecules and possess an unsaturated alpha-isoprene residue. Analogy with the role of bactoprenyl phosphates in the synthesis of bacterial wall glycans led to the discovery that the mono- and di-phosphates of dolichols function as cofactors in protein N-glycosylation, involving the formation of glycosylated derivatives of dolichol as intermediates. Variation of the concentration of dolichyl phosphate was shown to be one way of controlling protein N-glycosylation. This can be achieved by modification of the relative activities of dolichol kinase and dolichol phosphate phosphatase. Modulation of the biosynthetic pathway, still not fully understood, of dolichyl phosphate may also be an important factor. Several disease conditions involve aberrations in these pathways.

Characterization of the LDL receptor in rat promegakaryoblasts in culture.

Rat promegakaryoblasts (RPM, a precursor platelet cell line) in culture exhibited a capacity to bind, take up and degrade 125I-LDL. The low density lipoprotein (LDL) binding showed the following characteristics: (a) high affinity, (b) saturability, (c) specificity, (d) down-regulation, after exposure to 25 hydroxycholesterol. Furthermore the proteolytic degradation of 125I-LDL by RPMs was inhibited by chloroquine which interferes with the lysosomal degradation processes. These findings show LDL receptor cell biology of RPM to be of the classical type and to differ from that of platelets.

Changes in murine tissue concentrations of dolichol and dolichol derivatives associated with age.

The concentrations of the three major cellular forms of dolichol (free, esterified and phosphorylated) were determined in murine liver, kidney and heart. The tissue levels of these forms of dolichol were studied in detail as a function of age. Changes in the activities of dolichyl phosphate phosphatase and dolichol kinase were also determined. In liver, the concentration of unesterified dolichol, fatty acyl dolichol and dolichyl phosphate increased markedly over a period of 6 to 25 months (four-fold, 5.5-fold and nine-fold, respectively). In kidney only, free dolichol and phosphorylated dolichol increased (approximately four-fold in each case). However, this tissue consistently showed the highest concentrations of all forms of dolichol as compared to liver and heart. In heart, both free and esterified dolichol concentrations increased (approximately 3.25-fold in each case); dolichyl phosphate levels were not determined in this tissue. In all tissues studied, the activity of the dolichyl phosphate phosphatase enzyme was considerably higher than that of the dolichol kinase enzyme. In liver, there was no evidence to suggest that either enzyme was critical in determining the relative concentrations of dolichol and dolichyl phosphate. Evidence for such a role for the kinase in the kidney was stronger. Treatment of aging mice with meclofenoxate, a drug that is reported to cause dissolution of lipofuscin, failed to prevent accumulation of dolichol and dolichyl phosphate with age. These observations suggest that not all accumulated dolichol is associated with lipofuscin. Meclofenoxate treatment had no consistent effect on the activities of the enzymes studied.