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Four classes (pP_xGMⁱ to pP_xGM^iv) of extracellular peptidophosphogalactomannan (pP_xGM) (Fig. 1) with 'x' phosphodiester residues occur in cultures of Penicillium charlesii (renamed Penicillium fellutanum) (9). pP_xGMⁱ contains the largest percentage of phosphocholine phosphodiester residues attached to the mannan region and is eluted from DE-52 with 0.01 M HCl; elution of pP_xGM^ii-iii from DE-52 occurs with 0.01 N HCl-0.06 M LiCl and 0.01 N HCl-0.4 M LiCl, respectively. pP_xGM^iv has the fewest phosphocholine residues and is eluted with 0.5 M NaCl (9). pP_xGMⁱⁱ and pP_xGMⁱⁱⁱ represent about 80 and 5 mol%, respectively, of pP_xGM^ii-iv (10). All pP_xGMs contain galactofuranosyl-6-O-1'-O-[N-peptidyl-(2-aminoethanol)] residues with ³¹P signals at 1.15, 1.33, and 1.47 ppm (1, 9, 11, 12). pP_xGMⁱⁱ in day 4 P. fellutanum cultures in standard growth (SG) medium (9), (initially 20 mM phosphate) contains up to 60 phosphodiesters (pP₆₀GMⁱⁱ) (1, 2, 11); day 5 cultures contain pP₃₀GMⁱⁱ. Phosphodiester content decreases with culture maturation (9, 10). The data suggest that exocellular pP_xGM undergoes loss of phosphocholine phosphodiester units and that the affinity of a specific pP_xGM species depends on its affinity for DE-52.

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Structural features of peptidophosphogalactomannan. The structural features are based on methylation analyses and 13C and 31P NMR spectroscopy (1-3, 8, 11, 12).

The mass of serine, threonine, and glycine in pP_xGMⁱⁱ constitutes nearly 50% of the total amino acyl content of peptides in the polymer (3). Both the 3-kDa peptide to which the mannan is attached and galactose-6-O-phospho-1'-O-[N-peptidyl-(2'-aminoethanol)] phosphodiesters contain approximately the same percentage of glycine, serine, and threonine residues (1).

Earlier studies indicated that the extracellular pP_xGMs of P. fellutanum have a physiological role as reserve sources of specific nutrients which are released as needed by the fungus when nutrients such as phosphate, choline, and/or carbohydrate are limited (4, 5). In P. fellutanum cultured in phosphate-limited (LPSG) medium (9), the phosphocholine phosphodiester residues of pP_xGMⁱⁱ serve as precursors of choline-O-sulfate (COS) and glycine betaine (GB) (6). P. fellutanum is osmotolerant in an LPSG medium containing 3 M NaCl (7). The levels of the secondary osmoprotectants, GB and COS, increased 22- and 2.6-fold, respectively, in the mycelial cytoplasm of cultures in LPSG medium containing 3 M NaCl. This constitutes a response to osmotic stress on the organism that cannot be met by production of glycerol (7). Glycine, serine, and threonine in pP_xGM are potential precursors of COS and GB.

Our objective was to determine if stressing P. fellutanum with 3 M NaCl modifies extracellular acid phosphomonoesterases (AP), nonspecific phosphocholine:phosphodiesterase(s) (PC:PCH), and/or bis-(p-nitrophenol)-phosphate phosphodiesterase (Bis-PDase), activities associated with loss of phosphocholine and modification of phospho-1-O-[N-peptidyl-2-(aminoethanol)] phosphodiester residues in pP_xGMs. A preliminary report of some of this work has appeared elsewhere (1).

Influence of NaCl concentration on activity of AP and phosphodiesterases. P. fellutanum cultured in a medium (LPSG) containing 2 mM phosphate has high activities of AP, PC:PCH, and Bis-PDase compared with SG medium cultures (9). Nutrients and culture conditions for P. fellutanum have been previously described (5, 6). The substrates for AP, PC:PCH, and Bis-PDase activities were p-nitrophenyl-phosphate, p-nitro-phenylphosphoryl choline, and bis-(p-nitrophenyl)-phosphate, respectively. Sterile NaCl was added 72 h after inoculation of cultures of P. fellutanum in LPSG media. The LPSG cultures contained 0.01 (control), 0.51, 1.01, 2.01, and 3.01 M NaCl. Two to three milliliters of culture was removed daily through day 15. After filtration through Whatman no. 4 paper, a mixture containing 100 µl of filtrate, 500 µl of 10 mM sodium citrate buffer (pH 5.0), 2 µmol of substrate, and double-distilled water (to 850 µl) was incubated at 22°C for 20 min. Reactions were stopped with 1 ml of 0.2 N NaOH, and the amount of p-nitrophenoxide produced was determined at 410 nm. The experimental results of the assays were reproducible within 10% or less. The concentration of NaCl, up to 0.40 M, seemed to have little effect on the measured in vitro activity of PC:PCH (data not shown).

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Influence of NaCl concentration on the production of phosphoesterases in LPSG medium. Sterile solid NaCl was added to 150 ml of P. fellutanum cultures 72 h after inoculation of spore suspensions into LPSG medium to the concentration shown in each panel. Samples of culture were removed, filtered, and assayed (9) for AP, PC:PCH, and Bis-PDase activities daily through day 15. All activities were reproducible within 5%. Salt concentration did not influence the activity of PC:PCH.

Ratio of pP_xGMⁱⁱ to pP_xGMⁱⁱⁱ from LPSG cultures. The culture filtrates from day 8 P. fellutanum cultured on LPSG medium and on LPSG medium adjusted to 3.01 M NaCl at 72 h after inoculation were exhaustively dialyzed against water in 14-kDa molecular weight cutoff membranes. The pP_xGMs were concentrated by precipitation with Cetrimide and fractionated on DEAE-cellulose (Fig. 3) (5). Carbohydrate content of fractions was determined by a modified microphenol-sulfuric acid assay (2). Eighty percent or more of the pP_xGM from the LPSG control medium was obtained as pP_xGMⁱⁱ (peak I) and the remainder as pP_xGMⁱⁱⁱ (peak II) (Fig. 3A). This ratio of pP_xGMⁱⁱ to pP_xGMⁱⁱⁱ approximates that found previously (2, 10). In contrast, fractionation of pP_xGM from filtrates of cultures adjusted to 3.01 M NaCl resulted in a pP_xGMⁱⁱ/pP_xGMⁱⁱⁱ ratio of about 1:2 (Fig. 3B). The total mass of pP_xGMs recovered was about the same as that in controls. The data suggest that cultures in 3 M NaCl either convert pP_xGMⁱⁱ to pP_xGMⁱⁱⁱ or de novo synthesize significantly more pP_xGMⁱⁱⁱ and less pP_xGMⁱⁱ than controls.

View larger version (29K): [in this window] [in a new window]   FIG. 3.   Fractionation of pPxGMs from LPSG medium (A) and LPSG medium adjusted to contain 3.01 M NaCl (B). The cultures were filtered through Whatman no. 4 paper, and the pPxGMs were precipitated with Cetrimide as described previously (7) and fractionated on DEAE-cellulose (7). pPxGMii (peaks I) was eluted with 0.01 N HCl-0.06 M LiCl, and pPxGMiii (peaks II) was eluted with 0.01 N HCl-0.4 M LiCl.

³¹P NMR spectra of pP_xGMⁱⁱ and pP_xGMⁱⁱⁱ from LPSG cultures containing 3.01 M NaCl. The pP_xGMⁱⁱ (150 mg) from the control LPSG culture (Fig. 3A, peak I) and both pP_xGMⁱⁱ (Fig. 3B, peak I) and pP_xGMⁱⁱⁱ (Fig. 3B, peak II) from a culture containing 3.01 M NaCl were analyzed separately by proton-decoupled ³¹P nuclear magnetic resonance (NMR) spectroscopy. The spectrum of pP_xGMⁱⁱ from the control (Fig. 4A) is similar to that of LPSG cultures previously reported (2, 5, 9) with ³¹P signals at 0.22, 1.15, 1.33, and 1.47 ppm and minor signals of phospho-1-O-[2-aminethanol] (0.9 ppm), phospho-1-O-[N-methyl-2-aminoethanol] (0.8 ppm), and phospho-[N,N'-dimethyl-2-aminoethanol] (0.7 ppm). The relative heights of peaks b to d were comparable in pP_xGMⁱⁱ from both LPSG and SG medium, indicating little change in relative quantities of these phospho-1-O-[N-peptidyl-(2-aminoethanol)] phosphodiester species (1, 9).

View larger version (10K): [in this window] [in a new window]   FIG. 4.   31P NMR spectra of pPxGMii and pPxGMiii isolated from day 8 P. fellutanum cultured in LPSG medium containing 3.01 M NaCl. Proton-decoupled 31P NMR spectroscopy of 150 mg of pPxGMii from LPSG (A) and pPxGMii (B) and pPxGMiii (C) from LPSG cultures amended to contain 3.01 M NaCl at 72 h after inoculation was carried out. Data acquisition parameters for panel A are as follows: spectral width, 2,403.8 Hz; pulse width, 21 µs; acquisition time, 1.70 s. Spectrometer frequency was 121.4685 MHz; temperature was 23°C. Chemical shifts were assigned previously (1, 12): peak a, phosphocholine phosphodiester, 0.22 ppm; peaks b through d, phospho-1-O-[N-peptidyl-(2-aminoethanol)] phosphodiesters, 1.15, 1.33, and 1.47 ppm, respectively, attached to the mannopyranosyl-6-O- and galactofuranosyl-6-O- positions of pPxGMs.

Amino acid composition of peptidophosphomannan from cultures in LPSG medium containing 5 M NaCl. A peptidophosphomannan, pP_xM, was isolated from filtrates of P. fellutanum cultured for more than 20 days in LPSG medium containing 5 M NaCl. pP₂M eluted from DE-52 in 0.01 N HCl-0.06 M LiCl. This polymer was treated with 6 N HCl at 110°C for 24 h, and amino acid analyses were performed. Serine was the only amino acid found in the neutralized hydrolysate. Similar acid treatment of pP₃₀GMⁱⁱ isolated from day 5 P. fellutanum, cultured in SG medium containing 20 mM phosphate, showed that serine, threonine, and glycine, potential precursors of COS and GB, represent 46 mol% of the total amino acyl residues. pP₃₀GMⁱⁱ was treated with 0.01 N HCl at 100°C for 90 min to release galactofuranosyl residues. The hydrolysate was neutralized and fractionated by dialysis in a 1-kDa molecular weight cutoff membrane. The mole percent of glycine, serine, and threonine in the high-molecular-weight peptidophosphomannan in the retentate fraction was 47, compared with 42 in the low-molecular-weight galactofuranosyl-containing anionic fraction. This shows that both the peptidophosphomannan-region and galactofuranosyl-6-O-phospho-1'-O-[N-peptidyl-(2'-aminoethanol)]-containing galactofuran chains are potential sources of glycine, serine, and threonine.