Abstract
Cell wall transferases utilizing beta-(1-3)-glucan chains as substrates may play important roles in cell wall assembly and rearrangement, as beta-(1-3)-glucan is a major structural component of the cell wall of many fungi. A novel beta-(1-3)-glucanosyltransferase was purified to apparent homogenei ty from an autolysate of the cell wall of Aspergillus fumigatus. The enzyme had a molecular mass of 49 kDa and contained approximately 5 kDa of N-linked carbohydrate. The enzyme catalyzed an initial endo-type splitting of a beta-(1-3)-glucan molecule, followed by linkage of the newly generated reducing end to the nonreducing end of another beta-(1-3)-glucan molecule. Laminarioligosaccharides of size G10 and greater were donor substrates for the transferase. Laminarioligosaccharides of size G5 and greater formed acceptors. The enzyme was able to reuse initial transferase products as donors and acceptors in extended incubations, resulting in the formation of increasingly larger transferase products until they became insoluble. The major initial products from an incubation of the transferase with borohydride-reduced G11 (rG11) were rG6 and rG16. 1H NMR analysis of the rG16 transferase product showed it was a laminarioligosaccharide, indicating that the enzyme forms a beta-(1-3)-linkage during transfer. The enzyme may have a key function in vivo by allowing the integration of newly synthesized glucan into the wall and promoting cell wall expansion during cell growth.
Highlights
The fungal cell wall is a highly dynamic structure and despite decades of research elucidating its composition, very little is known about the processes involved in its assembly and subsequent rearrangements during cell growth
The assembly and rearrangement of -(1–3)-glucan once external to the plasma membrane must involve the coordinated action of many different cell wall enzymes, and these may play a direct role in events such as the integration of the newly synthesized -(1– 3)-glucan into the existing wall glucan, branching of -(1–3)glucan via -(1–3)/(1– 6)-branchpoints, as well as the crosslinking of -(1–3)-glucan to other wall components such as chitin or proteins
Cabib and coworkers (11) have shown that some of the cell wall chitin is linked through a -(1– 4)-linkage from the terminal residue to the nonreducing end of -(1–3)-glucan chains
Summary
Preparation of Cell Walls and Autolysis—A. fumigatus CBS 144 – 89 was grown in a 15-liter fermenter in 2% glucose, 1% mycopeptone (Biokar Diagnostics) plus 0.1% silicone antifoam 426R (Rhodorsil) at 25 °C, 500 rpm, 8 liters of air 1⁄7 minϪ1 for 42 h. Fractions containing the transferase activity were pooled, dialyzed against 10 mM -mercaptoethanol, 5 mM EDTA, 10 mM sodium acetate buffer, pH 4.0, and applied to a Mono S column (HR 5/5, Pharmacia) and eluted with a linear NaCl gradient (0 –300 mM in 40 min) at a flow rate of 0.8 ml minϪ1. The fractions containing the transferase activity were pooled, dialyzed against 10 mM Tris/HCl, pH 7.0, and deposited on a DEAE-5PW column (8 ϫ 75 mm, TosoHaas) and eluted with a linear NaCl gradient (0 –300 mM in 60 min) with a flow rate of 0.75 ml minϪ1. Fractions containing the transferase activity were pooled, dialyzed against 10 mM -mercaptoethanol, 5 mM EDTA, 10 mM sodium acetate, pH 4.0, and deposited on a CM-5PW column (8 ϫ 75 mm, TosoHaas) and eluted with a linear NaCl gradient (0 –300 mM in 60 min) at a flow rate of 0.8 ml minϪ1. De-Nglycosylation of glycoprotein was done using recombinant N-glycosidase F (Oxford GlycoSystems) according to the manufacturer’s instructions
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