FIGURE 1.

Skp1 modification pathway and AgtA expression constructs. A, schematic of the Skp1-hydroxylation/glycosylation pathway in Dictyostelium. The modification occurs on Pro-143 near the C terminus, and is initiated by the activity of the prolyl hydroxylase PhyA. Five glycosyltransferase reactions mediated by 3 proteins follow sequentially to yield a Hyp-linked linear pentasaccharide. B, diagram of full-length His₆AgtA and the His₆-tagged domain constructs expressed in E. coli and Dictyostelium. The N-terminal catalytic domain construct (His₆AgtA-CAT) terminates 33 amino acids after the end of sequence similarity (codon 274) to other CAZy GT77 sequences (2), and the C-terminal expression construct (His₆AgtA-WD40) initiates 19 amino acids upstream of this boundary. The TEV protease site inserted after the His₆ tag, the candidate canonical DXD motif, and the conserved His-268, are indicated. C, purified His₆AgtA-FL, His₆AgtA-CAT, and His₆AgtA-WD40 were analyzed by SDS-PAGE and staining with Coomassie Blue for total protein.