Volume 24, Number 6,
March 15, 2016
, Pages 1362-1368
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Abstract
For the first time, we describe the chemical synthesis of tetramannoside, which contains botha(1→2) e b (1→2) see. dodecyltium (lauryl)glycosideswere prepared from odorless dodecyl thiols and used as donors forglycosylationsteps. This tetramannoside, linked to the mantle group, proved to be a perfect substrate for the β-mannosyltransferase Bmt3, confirming the proposed specificity and allowing the preparation of the pentamannoside sequence (β Man (1,2) β Man (1,2) α Man (1,2) α Man (1,2) α Man) which can be used as a new substrate for further elongation studies.
graphic summary
Introduction
Candida albicans is a human pathogenic fungus involved in severe opportunistic infections. On the surface of its cell wall, it expresses β (1,2)-oligomannosides (β-Mans), which are crucial for yeast virulence mechanisms.1These β-Mans replace the terminal α-Man residues of various glycoconjugates, including polymannosylated N-linked glycans and the family of Candida-specific glycolipids, phospholipomannan (PLM). The structure of these mannoconjugates has been extensively studied in recent years, but their origin is still unclear. A family of nine genes (BMT) encoding β-mannosyltransferases (Bmt) was identified in this yeast using a mutant phenotypic approach.2Several of these Bmts are predicted to be involved in the biosynthesis of β-Mans from N-glycans and PLM.2,3 Recently, the activity and specificity of Bmt1 have been characterized using a recombinant form of the enzyme. It was shown that Bmt1 is responsible for the initiation of β-Mans in the acid-stable part of polymannosylated N-glycans. 4, 5 Now, the activity of Bmt3, an enzyme that is probably involved in the elongation of β-Mans N-glycans, needs to be investigated (Figure 1) .
In order to characterize the roles of β-mannosyl transferases, particularly their substrate requirements and their kinetics, fluorescently labeled synthetic oligomannosides are desirable. We recently published our work on Bmt14, 5 using coat-labeled α(1,2) oligomannosides as valuable substrates. In order to exploit the activity of the second enzyme Bmt3, an α(1,2) tetramannoside with a β unit at the non-reducing end was chosen as a suitable mimic of its natural substrate (Figure 2). No synthetic work on this mixture of α,β-oligomannosides has been reported in the literature.
Assembled1can also originate from enzymatic mannosylation of trimannosides using our recombinant Bmt1, chemical synthesis is preferred here as it offers larger amounts of a fully defined compound.
paragraphs of sections
the results
Synthesis of1is based on two carbohydrate building blocks (Figure 3): an α-mannosyl donor (2), equipped with a thiolauryl leaving group, and a similar β-glucopyranosyl donor (3). Azidopropanol (5) was chosen as the ligand, and methyl isatoic anhydride (4) as the mantle fluorophore precursor. The mantle group is a 'discrete' fluorophore, it is small, polar and uncharged at pH 7 and should not interfere with the enzymatic process. It is proven to be effective in Bmt1 work. (Excitation 334 nm, emission
enzymatic evaluation
Synthetic mantle-mannonetetraose DP4 1 ([M+Na]+at m/z 879.4) was used as an acceptor substrate to study the enzymatic properties of Bmt3p. The presumed function of this enzyme is to add a β-Man residue to α-1,2 oligomannosides capped with a non-reducing terminal β-1,2 Man unit. Recombinant soluble Bmt3p enzyme was produced in Pichia pastoris11and the enzymatic evaluation was carried out using GDP-Man as the mannosyl unit donor. The reaction products were monitored by normal phase HPLC
Conclusion
For the first time, we describe the chemical synthesis of tetramannoside, which contains both α (1,2) and β (1,2) linkages. This tetramannoside proved to be a perfect substrate for the enzyme Bmt3,11confirming the proposed specificity of the enzyme. The resulting pentamannoside will be useful as a new substrate for studies of elongation by other transferases such as Bmt4.
Enzymatic assay of mannosyl transferase
A culture supernatant of an engineered strain of Pichia pastoris containing about 35 mgL−1Bmt3p, 0.05% Tween 20 and 0.5% Triton X-100 were used as the enzyme source. Details of the genetic construction and culture conditions are described elsewhere.11Assay mixture containing 2 μL supernatant, 0.06 mM mantyl-tetramannoside (1), 20 mM GDP-Man donor, 50 mM sodium citrate buffer pH 6.5, 20 mM CaCl2, 0.3% Triton X-100, in a total volume of 25 μL, was incubated for 30 minutes at 28°C (standard assay). ON
Recognitions
This work was supported by the Agency Nationale de la Recherche (ANR MIE CaBMT), with a grant fromEuropean UnionALLFUN (FP7/2007 2013, HEALTH-2010-260338) (Fungi in the Environment of Inflammation, Allergy and Autoimmune Diseases: Translating Basic Science into Clinical Practice “ALLFUN” and fromMinistry of Higher Education and Science(za G.S.-L. i T.H.).
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Synthesis of a biotechnologically obtained biotinylated β-D-mannopyranosyl-(1→2)-D-mannopyranose probe for the assessment of carbohydrate specificity of antibodies
2019, Carbohydrate Research
Excerpt from the quote:
Acidic hydrolysis of phosphomannan followed by enzymatic dephosphorylation efficiently yielded the disaccharide β-d-mannopyranosyl-(1 → 2)-d-mannopyranose 2 [1]. The latter can be considered a precursor of the β-(1 → 2)-linked dimannoside block in the synthesis of oligosaccharide derivatives linked to mannan β-mannoside side chains of yeast [2] whose direct chemical synthesis is often a non-trivial task [3-5]. Here we describe the transformation of disaccharide 2 (Scheme 1) into the glycosyl donor 5 and subsequently into the biotinylated probe 9.
Disaharid β-d-manopiranosil-(1→2)-d-mannopyranose obtained by chemical cleavage and enzymatic dephosphorylation of biotechnologically available phosphomannan was transformed in six steps into a biotinylated probe suitable for assessing the carbohydrate specificity of antibodies induced by yeast cell wall preparations.
Synthesis of oligosaccharides related to polysaccharides of the cell wall of Candida and Aspergillus fungi
2017, Russian Chemical Reviews
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