Disulfide Connectivity of Velaglucerase

From Proteopedia

Human beta-glucocerebrosidase

Human beta-glucocerebrosidase, also known as glucoceremidase, GCase, and velaglucerase, is an enzyme that is commonly found to be deficient in patients with Gaucher's disease. Currently, there are three drugs on the market to treat this disease. One being Genzyme's Cerezyme, Protalix's Taliglucerase and SHIRE's VPRIV.

The typical method used to sequence current biologics, as well as map post translational modifications, is a combination of proteolytic digestion followed by LC/MS. Unfortunately, sometimes this method is not enough for complete identification of some post translational modifications. I am interested in the oxidation of Cys residues for the formation of disulfide bonds.

Disulfide Structure

Human beta-glucocerebrosidase (GCase) has seven Cys residues, four of which make two disulfide bonds. These two disufide bonds are both located in the N-terminal region of the protein and separated by only one amino acid. One of my current projects is using mass spectrometry to discern the disulfide connectivity in regions of a protein that are rich with Cys residues.

GCase N-terminal residues 1-23: ARPCIPKSFGYSSVVCVCNATYCDS

Disulfide Connectivity: C4-C16 & C18-C23

3D structures of beta-glucocerebrosidase

Acid-beta-glucosidase

Additional Resources

For additional information, see: Metabolic Disorders

Reference

• Dvir H, Harel M, McCarthy AA, Toker L, Silman I, Futerman AH, Sussman JL. X-ray structure of human acid-beta-glucosidase, the defective enzyme in Gaucher disease. EMBO Rep. 2003 Jul;4(7):704-9. PMID:12792654 doi:10.1038/sj.embor.embor873
• Shaaltiel Y, Bartfeld D, Hashmueli S, Baum G, Brill-Almon E, Galili G, Dym O, Boldin-Adamsky SA, Silman I, Sussman JL, Futerman AH, Aviezer D. Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher's disease using a plant cell system. Plant Biotechnol J. 2007 Sep;5(5):579-90. Epub 2007 May 24. PMID:17524049 doi:10.1111/j.1467-7652.2007.00263.x
• Brumshtein B, Salinas P, Peterson B, Chan V, Silman I, Sussman JL, Savickas PJ, Robinson GS, Futerman AH. Characterization of gene-activated human acid-beta-glucosidase: crystal structure, glycan composition, and internalization into macrophages. Glycobiology. 2010 Jan;20(1):24-32. Epub 2009 Sep 9. PMID:19741058 doi:10.1093/glycob/cwp138