Molecular-scale features that govern the effects of O-glycosylation on a carbohydrate-binding module

Abstract

Protein glycosylation is a ubiquitous post-translational modification in all kingdoms of life. Despite its importance in molecular and cellular biology, the molecular-level ramifications of O-glycosylation on biomolecular structure and function remain elusive. Here, we took a small model glycoprotein and changed the glycan structure and size, amino acid residues near the glycosylation site, and glycosidic linkage while monitoring any corresponding changes to physical stability and cellulose binding affinity. The results of this study reveal the collective importance of all the studied features in controlling the most pronounced effects of O-glycosylation in this system. Going forward, this study suggests the possibility of designing proteins with multiple improved properties by simultaneously varying the structures of O-glycans and amino acids local to the glycosylation site.

Fig. 1. The NMR structure of the Family 1 CBM and the top layer of cellulose. The tyrosine residues are shown in purple. The O-linked mannose at Ser3 site is shown in blue.

Fig. 2. Synthetic CBM isoforms and the structures of O-linked glycans.

Fig. 3. The contributions of amino acids to the effects of the Ser3 glycosylation on the proteolytic stability (half-life to thermolysin degradation), thermostability (melting temperatures measured by variable temperature CD), and binding affinity (K _ads values on BMCC) of the TrCel7A CBM. All error bars reported are standard deviations of data achieved from three separate trials. The structural feature of each isoform is implied by its name, i.e. CBMS3(Manα) representing the isoform containing a single mannose α-linked to Ser3, CBMQ2A + S3(Manα) representing the isoform containing a Gln-to-Ala mutation at position 2 and a single mannose α-linked to Ser3, and CBMS3hSer(Manα) representing the isoform containing a Ser-to-hSer mutation at position 3 and a single mannose α-linked to hSer3. # 53 °C. * No observable binding noted.

Fig. 4. The contributions of different glycans to the effects of Ser3 glycosylation on the proteolytic stability, thermostability, and binding affinity of the TrCel7A CBM. All error bars reported are standard deviations of data achieved from three separate trials. * No observable binding noted.

Fig. 5. Correlation of (A) the change in melting temperature (ΔT _m) and change in half-life during thermolysin degradation (Δt _1/2), and (B) the change in binding affinity upon glycosylation. Data points represent differences between CBM glyco-variants and their corresponding unglycosylated counterparts. The data for the CBM pairs 15/14 and 34/14 are not included in the plot because of their unique characteristics.