The oligosaccharyltransferase complex from pig liver: cDNA cloning, expression and functional characterisation

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Oligosaccharyltransferase (OST) is a crucial enzyme complex for protein glycosylation.
• Understanding the function of individual OST subunits is essential for elucidating the complex's mechanism.
• Previous studies have not fully characterized the roles of specific OST subunits in catalytic activity.

Purpose of the Study:

• To investigate the functional roles of pig OST subunits OST48, ribophorin I, and ribophorin II.
• To determine the contribution of these subunits to the catalytic activity of the OST complex.
• To analyze the subcellular localization and potential ER-retention signals of OST48 and ribophorin I.

Main Methods:

• Cloning and in vitro translation of OST48, ribophorin I, and ribophorin II cDNAs.
• Expression of these proteins in COS-1 cells and analysis of their molecular masses and topology.
• Assays for OST activity upon co-expression of different subunit combinations.
• Analysis of protein localization using immunofluorescence and manipulation of ER-retention signals.

Main Results:

• OST48, ribophorin I, and ribophorin II were characterized as type I membrane proteins localized to the ER lumen.
• Co-expression of OST48 and ribophorin I (or a myc-tagged variant) resulted in a modest increase in OST activity (approx. 25%).
• Neither OST48 nor ribophorin I exhibited OST activity when expressed alone.
• OST48 contains a double-lysine ER-retention motif, while ribophorin I does not.
• Interaction with ribophorin I, rather than the lysine motif, appears to mediate OST48's ER localization.

Conclusions:

• OST48 and ribophorin I are likely key catalytic subunits of the hetero-oligomeric OST complex.
• The limited increase in activity suggests potential instability or folding issues of subunits lacking other complex components.
• Ribophorin I plays a role in retaining OST48 within the endoplasmic reticulum.