Mannans

A neoglycoprotein (XXXG-BSA) was prepared by coupling a heptasaccharide containing 3 xylosyl and 4 glucosyl residues (XXXG, Megazyme, Bray, Ireland) to BSA by reductive amination [42 (link)]. XXXG (30 mg) was dissolved in 1.0 ml of 0.2 M sodium borate buffer pH 9.0. This was followed by the addition of 20 mg BSA and then 30 mg of sodium cyanoborohydride. The mixture was maintained in a water bath at 50°C with occasional mixing. After 24 h the pH was adjusted to pH 4.0 by the addition of 45 μl of 80% (v/v) acetic acid. The solution was then dialysed extensively against distilled water with several changes over 4 days.
Rat immunization, hybridoma preparation and cloning procedures were performed as described previously [34 (link)]. Two male Wistar rats were injected with 100 μg XXXG-BSA in complete Freund's adjuvant administered subcutaneously on day 0, with the same amount administered with incomplete Freund's adjuvant on days 33 and 71. On day 145, a selected rat was given a prefusion boost of 100 μg XXXG-BSA in 1 ml PBS by intraperitoneal injection. The spleen was isolated three days later for isolation of lymphocytes and fusion with rat myeloma cell line IR983F [43 ]. Antibodies were selected by ELISA using tamarind xyloglucan as antigen. Subsequent characterization was by means of a glycan microarray of cell wall polymers [28 (link)] and competitive inhibition ELISAs using the xyloglucan XXXG heptasaccharide from tamarind xyloglucan and a series of related xyloglucan oligosaccharides. A mixture of the XXLG and XLXG octasaccharide isomers and the XLLG nonasaccharide were derived from tamarind xyloglucan as described [44 (link)] and purified by HPLC using Tosoh TSK Gel Amide column (21.5 × 300 mm) eluted with 65% aqueous acetonitrile. Cellotetraose GGGG was prepared by acetolysis of cellulose [45 ] and separated from the mixture of deacetylated oligosaccharides by HPLC as above. The sample of pea xyloglucan was a gift from Marie-Christine Ralet (INRA, Nantes, France). ELISAs were carried out as described previously [6 (link)] and in all cases immobilised antigens were coated at 50 μg/ml. Mannan, tamarind xyloglucan polymers, isoprimeverose and xylose disaccharide were obtained from Megazyme, Bray, Ireland. The selected antibody, an IgG2c, was designated LM15.

The raw material was a mixture of 80% birch (Betula pendula) and 20% European beech (Fagus sylvatica) wood chips, provided by Södra Cell (Mörrum, Sweden). The composition of the raw material mixture, SF_{raw mat}, is presented in Table 1. The wood chips were size reduced using a knife mill (Retsch GmbH, Germany) that was fitted with a 20-mm screen and sieved to retrieve the 2–10-mm fraction.Table 1

Composition of the raw material and solid and liquid fractions after STEX pretreatment

	Raw material	HEX-treated material		STEX treated material
	SFraw mat (wt%)	SFHEX (wt%)	LFHEX (g/L)	SFSTEX (wt%)	LFSTEX (g/L)
Carbohydrates, thereof	67.6 ± 1.4	83.9 ± 0.1	na	67.1 ± 1.1	na
Glucan/glucose	39.4 ± 0.7	67.1 ± 0.1	0.4 ± 0.0	59.2 ± 0.7	2.6 ± 0.0
Xylan/xylose	22.2 ± 0.5	13.2 ± 0.0	10.1 ± 0.1	5.7 ± 0.0	43.4 ± 0.7
Arabinan/arabinose	0.6 ± 0.0	BDL	0.2 ± 0.0	BDL	0.7 ± 0.0
Galactan/galactose	1.8 ± 0.1	BDL	0.7 ± 0.0	BDL	2.2 ± 0.0
Mannan/mannose	3.7 ± 0.2	3.6 ± 0.0	0.5 ± 0.0	1.9 ± 0.0	5.7 ± 0.2
Lignin, thereof	27.2 ± 1.1	14.5 ± 0.1	na	30.4 ± 0.0	na
Acid-soluble	6.5 ± 0.0	3.4 ± 0.2	na	2.9 ± 0.1	na
Acid-insoluble	20.7 ± 1.0	11.1 ± 0.1	na	27.5 ± 0.1	na
Ash	BDL	BDL	na	BDL	na
Recovery	94.9 ± 2.5	98.4 ± 0.0	na	97.6 ± 1.1	na

Data represent mean values and standard deviation. Analyses were performed in duplicate

BDL below detection limit, na not applicable, SF solid fraction, LF liquid fraction, STEX steam explosion, HEX hydrotropic extraction

NMR experiments were recorded at 308 K either on an 800 MHz Avance NEO with an 18.8 Tesla magnetic field or on a 600 MHz Avance III HD spectrometer with a 14.1 Tesla magnetic field, both from Bruker (Billerica, MA, USA). Spectrometers were equipped with cryogenically cooled triple resonance ¹H[¹³C/¹⁵N] probes. Spectra were recorded using TopSpin 4.0.7 on the 800 MHz spectrometer or Topspin 3.6.1 for the 600 MHz spectrometer (Bruker Biospin). ¹H and ¹³C chemical shifts were referenced to external DSS (2,2-dimethyl-2-silapentane-5-sulfonate, sodium salt).
Polysaccharide samples (5 to 20 mg) were dissolved either in 200 µL or 300 µL of D₂O (99.97% ²H atoms, Eurisotop, Saclay, France) and placed in 3 mm tubes (Norell HT, Sigma-Aldrich) or 4 mm Shigemi tubes, depending on the amount of the sample. Resonance assignment, glycosidic bonds identification, and J coupling measurements were achieved from homonuclear ¹H-¹H COSY [22 (link)] and natural abundance heteronuclear ¹H-¹³C experiments: HSQC spectra recorded with or without decoupling [23 (link)], H2BC [24 (link)], HMBC [25 (link)], and HSQC-TOCSY [26 (link)] with a 200 ms mixing time. Monosaccharide residues were identified from the anomeric region; their anomeric configuration was established from the corresponding chemical shifts and the ¹J_H1,C1 coupling constant. ³J_H1,H2 coupling constants were measured from ¹H-¹H COSY experiments obtained with a resolution of 1 Hz. Glycosidic bonds were identified using HMBC experiments. The proportion of the different monosaccharide residues was estimated from the integrals of the anomeric peaks on the ¹H-¹³C-HSQC spectrum.
Stimulated echo diffusion experiments of the yet undescribed mannan (G3Man, see Results) were performed at 298°K with convection compensation and bipolar gradients [27 (link)]. These diffusion-ordered experiments (DOSY) were recorded with a diffusion delay of 140 ms; gradients were applied during 3.4 ms with 16 varying intensities and a total recycle delay of 3 s. Experiments were performed in triplicate. The diffusion coefficients were calculated with Topspin DOSY standard routines.
The apparent diffusion coefficient (d) of mannan for selected individual signals was obtained by fitting the integral (I) of selected signals to gaussian decays as a function of the gradient intensity (G): $$I=Io e^{-d{G}^2}$$
where Io represents the signal that is integral in the absence of gradients. Integral errors were estimated with five times the noise standard deviation. Fits were performed with Kaleidagraph™ 4.5 (Synergy software).

To obtain cell wall glycans, yeast from an exponential phase culture of C. albicans WT was harvested by centrifugation and washed three times with sterile deionized water. To remove N-linked glycans, the pellet was resuspended in 25 mL of a solution of 120 mM NaOAc and 500,000 U/mL endoglycosidase H (New England Biolabs) and incubated overnight at 37 °C without shaking. After this time, the cells were harvested by centrifugation at 2000× g for 5 min, the supernatant containing the N-linked glycans was collected, and the pH was adjusted to 7.0 with 100 mM NaOH. The cell package was kept frozen at −20 °C for interaction studies [31 ]. O-linked glycans were obtained by β-elimination by resuspending the cell pellet in 10 mL of 100 mM NaOH and were incubated overnight at room temperature (25 °C) with shaking at 30 rpm. The samples were centrifuged at 2000× g, the supernatant was collected, and the pH was adjusted to 7.0 with 100 mM HCl. The cell package was kept frozen at −20 °C [32 (link)]. The glycans recovered in the supernatants were purified separately by adding an equal volume of Fehling’s reagent and subjected to heating to precipitate mannan, creating a mannan-copper complex. The supernatant was decanted, and 8 mL of 3M HCl was added. The solution was poured into 100 mL of methanol-acetic acid 8:1 (v/v) until the liquid turned green and allowed to stand at room temperature for several hours until sedimentation of the precipitate. The liquid was filtered, and the precipitate was recovered and dissolved again with methanol-acetic acid 8:1 (v/v). The dissolution and precipitation procedures were repeated until the methanol-acetic acid mixture stopped turning green. Subsequently, the mixture was washed with methanol and ethyl ether and allowed to dry at room temperature. Finally, the mannan sediment was separated by filtration and dissolved in distilled water [33 (link)]. The purified mannans were determined for protein content by the Bradford method, where a protein concentration of less than 1 mg/mL was considered acceptable for use. The mannans were placed in pre-weighed tubes to be lyophilized, then their dry weight was determined, and they were resuspended in K-H solution to a final concentration equivalent to 10 mM mannose. Before perfusion, the glycans were sonicated to eliminate possible aggregates, as already described for the cell walls.