Two homologues encoding human UDP-glucose:glycoprotein glucosyltransferase differ in mRNA expression and enzymatic...

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• UDP-glucose:glycoprotein glucosyltransferase (UGT) is crucial for endoplasmic reticulum (ER) quality control, preventing misfolded glycoproteins from exiting.
• Understanding UGT function requires identifying and characterizing its human homologues.

Purpose of the Study:

• To isolate and characterize human UDP-glucose:glycoprotein glucosyltransferase homologues.
• To investigate the functional differences and expression patterns of HUGT1 and HUGT2.

Main Methods:

• Isolation of cDNAs encoding human UGT homologues (HUGT1 and HUGT2).
• Expression of cDNAs in COS-1 cells and localization to the ER.
• Enzymatic assays to measure glucosyltransferase activity.
• Site-directed mutagenesis to identify essential catalytic residues.

Main Results:

• HUGT1 and HUGT2 were identified, sharing 55% sequence identity and containing ER retrieval signals.
• HUGT1 expression is induced by ER stress, while HUGT2 is not.
• Recombinant HUGT1 demonstrated significant glucosyltransferase activity, whereas HUGT2 did not under optimized conditions.
• Four essential catalytic residues in HUGT1 were identified through mutagenesis.

Conclusions:

• HUGT1 plays a critical role in ER quality control and is regulated by protein folding status.
• HUGT2 appears to be a non-functional homologue or functions under different conditions.
• Specific residues in HUGT1 are essential for its catalytic activity in glycoprotein processing.