# Xylosyltransferase engineering to manipulate proteoglycans in mammalian cells

## TL;DR

Scientists engineered enzymes to control proteoglycan production in mammalian cells, enabling better study and manipulation of these important cell surface structures.

## Contribution

A bump-and-hole strategy was developed to specifically tag and study proteoglycan glycosylation in mammalian cells.

## Key findings

- Engineered XT1/2 enzymes transfer a chemically tagged xylose analog to target proteins.
- The method allows profiling and modifying proteoglycans with defined glycan chains.
- The approach enables orthogonal analysis of proteoglycan biology using biochemical techniques.

## Abstract

Mammalian cells receive signaling instructions through interactions on their surfaces. Proteoglycans are critical to these interactions, carrying long glycosaminoglycans that recruit signaling molecules. Biosynthetic redundancy in the first glycosylation step by two xylosyltransferases XT1/2 complicates annotation of proteoglycans. Here we develop a chemical genetic strategy that manipulates the glycan attachment site of cellular proteoglycans. Through a bump-and-hole tactic, we engineer the two isoenzymes XT1 and XT2 to specifically transfer the chemically tagged xylose analog 6AzGlc to target proteins. The tag contains a bioorthogonal functionality, allowing to visualize and profile target proteins in mammalian cells. Unlike xylose analogs, 6AzGlc is amenable to cellular nucleotide-sugar biosynthesis, establishing the XT1/2 bump-and-hole tactic in cells. The approach allows pinpointing glycosylation sites by mass spectrometry and exploiting the chemical handle to manufacture proteoglycans with defined glycosaminoglycan chains for cellular applications. Engineered XT enzymes permit an orthogonal view into proteoglycan biology through conventional techniques in biochemistry.

The xylosyltransferase isoenzymes XT1 and XT2 catalyze the first glycosylation step in the biosynthesis of proteoglycans. Now, bump-and-hole engineering of XT1 and XT2 enables substrate profiling and modification of proteins as designer proteoglycans to modulate cellular behavior.