Characterization of the Neurospora crassa GH72 family of Laminarin/Lichenin transferases and their roles in cell wall biogenesis

Abstract

In Neurospora crassa vegetative hyphae, chitin, β-1,3-glucan (laminarin), and a mixed β-1,3-/β-1,4-glucan (lichenin) are the major cell wall polysaccharides. GH72 enzymes have been shown to function as β-1,3-glucanases and glucanosyltransferases and can function in crosslinking β-1,3-glucans together. To characterize the enzymatic activities of the N. crassa enzymes, we expressed GEL-1 with a HIS6 tag in N. crassa. A chimeric maltose binding protein:GEL-2 was produced in E. coli. Purified GEL-1 and GEL-2 were used to characterize their enzymatic activities. We employed thin-layer chromatography (TLC) and polyacrylamide carbohydrate gel electrophoresis (PACE) assays to visualize GEL-1 and GEL-2 hydrolase and transferase activities on lichenin and laminarin substrates. We determined that GEL-1 functions as a laminarinase (β-1,3-glucanase) and as a laminarin transferase. We found that GEL-2 can function as a laminarinase and as a licheninase (β-1,3-/β-1,4-mixed-glucanase) and can crosslink β-1,3-glucans together. We demonstrated that GEL-2 can form enzyme:lichenin intermediates, providing evidence that GEL-2 functions as a lichenin transferase as well as a β-1,3-glucan transferase and crosslinks both types of polysaccharides into the N. crassa cell wall.

Keywords: Fungal cell wall; GH72 family; Laminarin transferase; Lichenin transferase; Neurospora crassa.

Fig. 1

Phenotype of ∆gel-1, ∆gel-2, ∆gel-5 mutant. Panel A. Wildtype and mutant conidia were placed in the middle of Petri dishes containing Vogel's sucrose agar growth medium and allowed to grow for 2 days. Panel B. The growing edges of wildtype and mutant colonies were photographed after 12 h of growth on Vogel's sucrose agar. The hyphae from the mutant are unable to extend across the medium. Scale bar represents 1 mm.

Fig. 2

Purification profile of GEL-1 using Ni-NTA column. Panel A shows a Coomassie-stained gel of the purification and Panel B shows a western blot using antibody directed against the HIS6 tag. Lane 1: Cell lysate from control cells not expressing a HIS6-tagged protein. Lane 2: MW markers. Lane 3: cell extract containing GEL-1:HIS6. Lane 4: unbound protein after passage through the affinity column. Lanes 5–10: Elution fractions 2, 6, 9, 10, 14 and 18, respectively. GEL-1:HIS6 was eluted at the expected size of ∼65 kDa (arrow) and elution fraction 6 was used for enzymatic studies.

Fig. 3

Thin layer chromatography (TLC) analysis demonstrating the hydrolase and transferase activities of purified GEL-1. Lanes 1 and 2 contain laminaritriose without enzyme (lane 1) and with GEL-1 (lane 2). Lanes 3 and 4 contain laminaritetraose without (lane 3) and with GEL-1 (lane 4). Lanes 5 and 6 contain lamarinipentaose without (lane 5) and with GEL-1 (lane 6). Lanes 7 and 8 contain laminarihexaose without (lane 7) and with GEL-1 (lane 8). Lane 9 contains ladder laminarinoses (biose to hexaose). Lanes 10 and 11 contain laminarin without enzyme (lane 10) and with GEL-1 (lane 11). Lanes 12 and 13 contain lichenin without enzyme (lane 12) and with GEL-1 (lane 13). Lane 14 contains GEL-1 enzyme without polysaccharides. Blue arrows indicate hydrolytic products, and the black arrows indicate transferase products after enzymatic action by the purified enzyme. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Hydrolase activity of GEL-1 on laminarihexaose (1 mg/ml) using polyacrylamide carbohydrate gel electrophoresis (PACE). Lane 1 contains laminarinose ladder. Lane 2 contains the undigested laminarihexoase prior to the addition of GEL-1 (0 h). Lane 3 shows the presence of digestion products from GEL-1 enzyme activity (24 h). Black arrows indicate the hydrolytic products due to enzymatic action of GEL-1 purified protein.

Fig. 5

Thin layer chromatography (TLC) analysis of products generated by digestion of ΔGH72 polysaccharides by purified GEL-1. Lane 1 shows maltodextrin marker (MD). Lane 2 contains undigested polysaccharides prior to the addition of GEL-1 (Control/0 h). Lane 3 shows the small oligosaccharide digestion products (Assay/24 h). The arrows point to the digestion products.

Fig. 6

Time-course assay for purified GEL-1 enzymatic activity on laminarihexaose from 0 h to 24 h indicating that the hydrolase activity of GEL-1 can be seen as soon as 2 h and transferase activity can be detected after 8 h of incubation. LD: ladder laminarinoses (biose, triose, tetraose, pentaose and hexaose). 0 h (prior to addition of enzyme) through 24 h: Assay timepoints. Blue arrows indicate hydrolase activity, and the black arrow indicates transferase activity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Thin layer chromatography (TLC) analysis demonstrating the hydrolase and transferase activities of purified GEL-2. Lanes 1 and 2 contain laminaritriose without enzyme (lane 1) and with GEL-2 (lane 2). Lanes 3 and 4 contain laminaritetraose without (lane 3) and with GEL-2 (lane 4). Lanes 5 and 6 contain lamarinipentaose without (lane 5) and with GEL-2 (lane 6). Lanes 7 and 8 contain laminarihexaose without (lane 7) and with GEL-2 (lane 8). Lane 9 contains ladder laminarinoses (biose to hexaose). Lanes 10 and 11 contain laminarin without enzyme (lane 10) and with GEL-2 (lane 11). Lanes 12 and 13 contain lichenin without enzyme (lane 12) and with GEL-2 (lane 13). Lane 14 contains GEL-2 enzyme without polysaccharides. Blue arrows indicate hydrolytic products, and the black arrows indicate transferase products after enzymatic action by the purified enzyme. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Western blot analysis of purified MBP:GEL-2 when incubated with lichenin, laminarin and polysaccharides from ΔGH72 medium indicating the presence of enzyme:lichenin and enzyme:laminarin intermediates. Panel A: Western blot analysis of GEL-2:lichenin intermediate. Lane 1: control lane having MBP:GEL-2 without added polysaccharide. Lane 2: MBP:GEL-2 with ΔGH72 polysaccharides. Lane 3: MBP:GEL2 with lichenin (1 mg/ml). Panel B: Western blot analysis of GEL-2:laminarin intermediate. Lane 1: control lane having MBP:GEL-2 without polysaccharides. Lane 2: MBP:GEL-2 with ΔGH72 polysaccharides. Lane 3: MBP:GEL-2 with laminarin (1 mg/ml). The western blots were done with anti-lichenin (panel A) and anti-laminarin antibodies (panel B).

Supplementary Fig. S1

Amino acid sequences for native GEL-1 and HIS6-tagged GEL-1.

Supplementary Fig. S2

GEL-1 nucleotide sequences for HIS6-tagged GEL-1 (NCU08909).