Sigfrid Svensson

Characterization of the extracellular, water-insoluble α-D-glucans of oral streptococci by methylation analysis, and by enzymic synthesis and degradation☆

Abstract Methylation analysis of water-insoluble α- D -glucans synthesized from sucrose by culture filtrates from several strains of Streptococcus spp. has proved that all of the glucans were highly branched and that the chains contained (1→6)- and (1→3)-linked D -glucose residues not involved in branch points. Hydrolysis of the glucans with a specific endo-(1→3)-α- D -glucanase demonstrated that the majority of the (1→3)-linked glucose residues were arranged in sequences. D -Glucose was the major product of the hydrolysis, and a small proportion of nigerose was also released. The use of a specific endo-(1→6)-α- D -glucanase similarly indicated that the glucans also contained sequences of (1→6)-linked α- D -glucose residues, and that those chains were branched. Two D -glucosyltransferases (GTF-S and GTF-I), which reacted with sucrose to synthesize a soluble glucan and a water-insoluble glucan, respectively, were separated from culture filtrates of S. mutans OMZ176. The soluble glucan was characterized as a branched (1→6)-α- D -glucan, whereas the insoluble one was a relatively linear (1→3)-α- D -glucan. The hypothesis is advanced that the glucosyltransferases can transfer glucan sequences by means of acceptor reactions similar to those proposed by Robyt for dextransucrase, leading to the synthesis of a highly branched glucan containing both types of chain. The resulting structure is consistent with the evidence obtained from methylation analysis and enzymic degradations, and explains the synergy displayed when the two D -glucosyltransferases interact with sucrose. Variations in one basic structure can account for the characteristics of water-insoluble glucans from S. sanguis and S. salivarius, and for the strain-dependent diversity of S. mutans glucans.

Synthesis of the disaccharide 6-O-β-d-galactopyranosyl-2-acetamido-2-deoxy-d-galactose using immobilized β-galactosidase

Abstract The disaccharide 6-O-β- d -galactopyranosyl-2-acetamido-2-deoxy- d -galactose has been synthesized by transfer of the β- d -galactopyranosyl residue from lactose to 2-acetamido-2-deoxy- d -galactose utilizing the transferase activity of β-galactosidase from E. coli . To make the enzyme reusable, it was applied in an immobilized form covalently bound to Sepharose CL-4B. The yield of the disaccharide was about 20%, calculated on the amount of acetamido-deoxy- d -galactose added. The disaccharide could also be obtained by reversal of the hydrolytic activity of the enzyme, using d -galactose and 2-acetamido-2-deoxy- d -galactose as substrate. The yield in this reaction, however, was only 2–3 % under the conditions applied.

Synthesis of Galβ1-3GlcNAc and Galβ1-3GlcNAcβ-SEt by an enzymatic method comprising the sequential use of β-galactosidases from bovine testes andEscherichia coli

Galβ1-3GlcNAc (1) and Galβ1-3GlcNAcβ-SEt (2) were synthesized on a 100 mg scale by the transgalactosylation reaction of bovine testes β-galactosidase with lactose as donor andN-acetylglucosamine and GlcNAcβ-SEt as acceptors. In both cases the product mixtures contained unwanted isomers and were treated with β-galactosidase fromEscherichia coli which has a different specificity, under conditions favouring hydrolysis, yielding besides the desired products, monosaccharides and traces of trisaccharides. The products were purified to >95% by gel filtration, with a final yield of 12% of 1 and 17% of 2, based on added acceptor. In a separate experiment Galβ1-6GlcNAcβ-SEt (3) was synthesized by the transglycosylation reaction using β-galactosidase fromEscherichia coli. No other isomers were detected. Compound 3 was purified by HPLC.

A new method for degradation of the protein part of glycoproteins: isolation of the carbohydrate chains of asialofetuin

A new method has been developed for degrading the protein part of several glycoproteins, whilst leaving the carbohydrate portion virtually intact apart from partial degradation at the reducing end. The method is based upon stabilization of the glycosidic linkages of the sugar residues by trifluoroacetyl groups and subsequent cleavage of the peptide bonds by transamidation. The two reactions are carried out in a mixture of trifluoroacetic anhydride and trifluoroacetic acid. After O- and N-detrifluoroacetylation, the carbohydrate portion can be isolated and re-N-acetylated. The applicability of the method is demonstrated by the isolation from asialofetuin of the carbohydrate chains that are attached by N- and O-glycosyl links.

Synthesis of mannose oligosaccharides via reversal of the α-mannosidase reaction

SummaryThree naturally occurring isomers of the disaccharideO-α-d-mannosyl-d-mannoside were synthesized by reversing the hydrolytic activity of jack bean α-mannosidase at 75°C in a very high concentration of mannose. Higher oligosaccharides were also obtained at the later stages of the reaction. The maximum total yield of disaccharides was 37% (w/w) based on the total amount of saccharides.

Studies of the reversed α-mannosidase reaction in high concentrations of mannose

Abstract The reversal of the hydrolytic activity of α-mannosidase from jack bean has been studied in high concentrations of mannose. From an initial concentration of 85% mannose (w/w), a maximum yield of 70% (w/w) di- and oligosaccharides was obtained. The products were separated by gel chromatography and high-performance liquid chromatography, and characterized by gas-liquid chromatographymass spectrometry and 1 H-nuclear magnetic resonance spectroscopy. The equilibrium for the formation of di- and oligosaccharides was investigated, and the equilibrium constant for the formation of disaccharides was calculated to be 5.5 ± 0.9. The stability of the enzyme and the rate of formation of disaccharides were studied as functions of temperature, pH, and starting concentration of mannose. It was shown that the enzyme was not inhibited by a total sugar concentration of 83% (w/w), compared with 40%.

Characterization of a penta- and an octasaccharide from urine of a patient with juvenile GM1-gangliosidosis

Abstract Two structurally related oligosaccharides have been isolated from the urine of a patient with GM 1 -gangliosidosis type 2. The isolation procedure included ultrafiltration, gel chromatography on Sephadex G-25, and preparative paper chromatography. From structural studies including optical rotation, sugar analysis, methylation analysis, and chromium trioxide oxidation, the following structures are deduced: The octasaccharide has previously been reported to be present in both liver and urine of patients with GM 1 -gangliosidosis type 1 and type 2. The pentasaccharide is a new compound and is an integral part of the octasaccharide. The yields of the octasaccharide and pentasaccharide were 17 and 8 mg/liter of urine, respectively. Both compounds are most probably degradation products derived from the core of glycoprotein carbohydrate chains.

Increased urinary excretion of a glucose-containing tetrasaccharide in patients with duchenne muscular dystrophy

Duchenne muscular dystrophy is inherited as a sex-linked recessive trait. The primary cause of the disease is unknown, but several hypotheses exist, suggesting myopathic [1], neuronal [2], vascular [3], and auto-immune [4] mechanisms. Evidence in favour of abnormal functioning of cellular membranes has been put forward and also that this membrane defect may be generalized [5-7] . Several reports about structural alterations of membranes [8-10] , alterations of different membrane enzymes [11-13], and other membrane proteins [ 13-15] have appeared. Elevated levels of muscle enzyme activities (e.g., creatine kinase in serum) and their depletion in the muscle tissue reflect a leakage in the muscle-cell membrane. Urinary excretion of various peptides in Duchenne muscular dystrophy was described in [16]. It was also observed that some of these peptide fractions differed in their ability to affect the growth of chick embryomuscle fibroblasts in culture as compared with similar fractions from urine of normal individuals [16,17]. Urinary oligosaccharide excretion has been studied under different physiological conditions and in patients with various diseases [18]. It was recently reported that urine of a patient with glycogen-storage disease type II (Pompes disease) had a dramatically increased excretion of a glucose-containing tetrasaccharide (89.6 rag/24 h urine) [19,20]: a-D-Glc-(I~6)-a-D-Glc(I~4)-a-D-Glc-(1--~4)-D-Glc4, referred to here as (GIc)4. This oligosaccharide, which has been regarded as a limit oligosaccharide in the extracellular breakdown of glycogen, is present in small amounts in normal urine. Greatly increased excretion of the oligosaccharide was also observed in several cases with glycogenstorage disease type III (9.4-45.2 mg/24 h urine) [20]. Since muscular dystrophy is a predominant symptom in Pompes disease it was decided to investigate some individuals with muscular dystrophies of different etiology. A moderately increased excretion was found in two patients with Duchenne muscular dystrophy and in three patients with unclassified muscular disease [20]. This investigation has now been extended to include 20 Japanese patients of different ages with Duchenne muscular dystrophy of varying severity.

Isolation and characterization of a blood group A-specific urinary tetrasaccharide.

Blood group A-, Band H-active tetraen pentasaccharides have been isolated from urine of starved ABH secretors [ 1,2]. Ingestion of free galactose or a glycoside of galactose (lactose) induces the formation and excretion of blood group specific diand trisaccharides [3,4] and small amounts of hexaand heptasaccharides [4]. The serological activities of some Aand B-specific oligosaccharides have been studied and compared with those of oligosaccharides from soluble blood group A and B substances [3,5]. When urine from individuals of different ABO blood group and secretor status was analyzed by gas-liquid chromatography-mass spectrometry (GLC-MS) for the content of various oligosaccharides, a new component was discovered in some, but not in all, urines of blood group A secretors. We now report the isolation, characterization and serological characterization of this new component.

Isolation and characterisation of 4-O-β-D-mannopyranosyl-2-acetamido-2-deoxy-D-glucose from the urine of a patient with mucolipidosis II and its occurrence in normal and pathological urine

A number of neutral oligosaccharides have been isolated from human urine. The excretion of some of these oligosaccharides depends on the donor’s blood group [l-3], dietary status [ 2,4], or some physiological [5-71 and pathological [ 1,2,8-151 conditions. Patients with lysosomal storage diseases in which the degradation of glycoproteins is affected excrete abnormally large quantities of oligosaccharides which are presumably the substrates of the defective enzymes. Mucolipidosis II (Icell disease, ML II) and mucolipidosis III (ML III) probably result from defective post-translational processing of lysosomal enzymes in certain cells [ 16,171, which cause a multiple lysosomal hydrolase deficiency and increased excretion of sialic acidcontaining oligosaccharides [ 111. Apart from the sialylated oligosaccharides there has been no report of increased amounts of neutral oligosaccharides in urine from these patients. However, routine gas chromatography-mass spectrometry (GLC-MS) screening of human urinary oligosaccharides showed that a hitherto undescribed neutral disaccharide is also excreted by patients with ML II and ML III and to a lesser extent by normal individuals and patients with other lysosomal storage disorders. We now describe the isolation and structure of this disaccharide.