GSBS Student Publications

Student Author(s)

Heidi Malaby

GSBS Program

Biochemistry & Molecular Pharmacology

UMMS Affiliation

Department of Biochemistry and Molecular Pharmacology; Program in Neuroscience; Program in Chemical Biology

Date

8-5-2014

Document Type

Article

Medical Subject Headings

Animals; CHO Cells; Consensus Sequence; Cricetinae; Cricetulus; Glycopeptides; Glycosylation; Humans; Hydrophobic and Hydrophilic Interactions; Peptide Chain Initiation, Translational; Protein Binding; *Protein Biosynthesis; Protein Folding

Disciplines

Biochemistry | Molecular Biology

Abstract

Asparagine (N)-linked glycosylation is essential for efficient protein folding in the endoplasmic reticulum (ER) and anterograde trafficking through the secretory pathway. N-Glycans are attached to nascent polypeptides at consensus sites, N-X-T/S (X not equal P), by one of two enzymatic isoforms of the oligosaccharyltransferase (OST), STT3A or STT3B. Here, we examined the effect of the consensus site X and hydroxyl residue on the distributions of co- and post-translational N-glycosylation of a type I transmembrane glycopeptide scaffold. Using rapid radioactive pulse-chase experiments to resolve co-translational (STT3A) and post-translational (STT3B) events, we determined that NXS consensus sites containing large hydrophobic and negatively charged middle residues are frequently skipped by STT3A during protein translation. Post-translational modification of the cotranslationally skipped sites by STT3B was similarly hindered by the middle X residue, resulting in hypoglycosylation of NXS sites containing large hydrophobic and negatively charged side chains. In contrast, NXT consensus sites (barring NWT) were efficiently modified by the cotranslational machinery, reducing STT3B's role in modifying consensus sites skipped during protein translation. A strong correlation between cotranslational N-glycosylation efficiency and the rate of post-translational N-glycosylation was determined, showing that the OST STT3A and STT3B isoforms are similarly influenced by the hydroxyl and middle X consensus site residues. Substituting various middle X residues into an OST eubacterial homologous structure revealed that small and polar consensus site X residues fit well in the peptide binding site whereas large hydrophobic and negatively charged residues were harder to accommodate, indicating conserved enzymatic mechanisms for the mammalian OST isoforms.

Rights and Permissions

Citation: Biochemistry. 2014 Aug 5;53(30):4884-93. doi: 10.1021/bi500681p. Epub 2014 Jul 25. Link to article on publisher's site

Comments

ACS AuthorChoice - This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

Related Resources

Link to Article in PubMed

Journal Title

Biochemistry

PubMed ID

25029371

Share

COinS
 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.