Candida albicans β-1,2 mannosyl transferase Bmt3: Preparation and evaluation of the fluorescent substrate β(1,2), α(1,2)-tetramannosyl (2023)

Volume 24, Number 6,

March 15, 2016

, Pages 1362-1368

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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

Candida albicans β-1,2 mannosyl transferase Bmt3: Preparation and evaluation of the fluorescent substrate β(1,2), α(1,2)-tetramannosyl (3)
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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


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


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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