Specificity of O-Glycosylation in Enhancing the Stability and Cellulose Binding Affinity of Family 1 Carbohydrate-Binding Modules

Liqun Chen, Matthew R. Drake, Michael G. Resch, Eric R. Greene, Michael E. Himmel, Patrick K. Chaffey, Gregg T. Beckham, Zhongping Tan

Research output: Contribution to journalArticlepeer-review

82 Scopus Citations


The majority of biological turnover of lignocellulosic biomass in nature is conducted by fungi, which commonly use Family 1 carbohydrate-binding modules (CBMs) for targeting enzymes to cellulose. Family 1 CBMs are glycosylated, but the effects of glycosylation on CBM function remain unknown. Here, the effects of O-mannosylation are examined on the Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase at three glycosyla-tion sites. To enable this work, a procedure to synthesize glycosy-lated Family 1 CBMs was developed. Subsequently, a library of 20 CBMs was synthesized with mono-, di-, or trisaccharides at each site for comparison of binding affinity, proteolytic stability, and thermostability. The results show that, although CBM mannosyla-tion does not induce major conformational changes, it can increase the thermolysin cleavage resistance up to 50-fold depending on the number of mannose units on the CBM and the attachment site. O-Mannosylation also increases the thermostability of CBM glyco-forms up to 16 °C, and a mannose disaccharide at Ser3 seems to have the largest themostabilizing effect. Interestingly, the glyco-forms with small glycans at each site displayed higher binding affinities for crystalline cellulose, and the glycoform with a single mannose at each of three positions conferred the highest affinity enhancement of 7.4-fold. Overall, by combining chemical glycopro-tein synthesis and functional studies, we show that specific glyco-sylation events confer multiple beneficial properties on Family 1 CBMs.

Original languageAmerican English
Pages (from-to)7612-7617
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number21
StatePublished - 27 May 2014

NREL Publication Number

  • NREL/JA-5100-62284


  • Biofuels
  • Cellulase
  • Chemical synthesis
  • Protein engineering


Dive into the research topics of 'Specificity of O-Glycosylation in Enhancing the Stability and Cellulose Binding Affinity of Family 1 Carbohydrate-Binding Modules'. Together they form a unique fingerprint.

Cite this