Specificity of a Soluble UDP-Galactose:Fucoside α1,3-Galactosyltransferase That Modifies the Cytoplasmic Glycoprotein Skp1 in Dictyostelium

Abstract

Skp1 is an adaptor-like protein in E3SCF -ubiquitin ligases and other multiprotein complexes of the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by an unusual pentasaccharide containing a Gal␣1-Fuc linkage, whose formation is examined here. A cytosolic extract from Dictyostelium was found to yield, after 2400-fold purification, an activity that could transfer Gal from UDP-Gal to both a Fuc-terminated glycoform of Skp1 and synthetic Fuc conjugates in the presence of Mn 2؉ and dithiothreitol. The microsomal fraction was devoid of activity. The linkage formed was Gal␣1,3Fuc based on co-chromatography with only this synthetic isomer conjugate, and sensitivity to ␣1,3/6-galactosidase. Skp1 exhibited an almost 1000-fold lower K m and 35-fold higher V max compared with a simple ␣-fucoside, but this advantage was abolished by denaturation or alkylation of Cys residues. A comparison of a complete series of synthetic glycosides representing the non-reducing terminal mono-, di-, and trisaccharides of Skp1 revealed, surprisingly, that the disaccharide is most active owing primarily to a V max advantage, but still much less active than Skp1 itself because of a K m difference. These findings indicate that ␣-GalT1 is a cytoplasmic enzyme whose modification of Skp1 requires proper presentation of the terminal acceptor disaccharide by a folded Skp1 polypeptide, which correlates with previous evidence that the Gal␣1,3Fuc linkage is deficient in expressed mutant Skp1 proteins.

Arabidopsis thaliana RGXT1 and RGXT2 Encode Golgi-Localized (1,3)-α-d-Xylosyltransferases Involved in the Synthesis of Pectic Rhamnogalacturonan-II

“…Another possible explanation regards the conformation of the growing A-chain, which may be fixed during synthesis in another conformation than the preferred conformation of the free disaccharide, which would thus encounter steric hindrance in the active site that methyl a-L-Fuc would not experience. Ketcham et al (2004) made analogous observations with the CAZy GTfamily-77 D. discoideum galactosyltransferase, where a trisaccharide substrate analog was inferior as acceptor compared with the corresponding disaccharide and the a-L-Fuc methyl glycoside acceptor. The authors propose that the fucosyl residue folds back in the trisaccharide but is prevented from doing so in the native glycoprotein acceptor, hence rendering the trisaccharide a poorer than expected acceptor analog.…”

“…RGXT1 and RGXT2 are novel and do not appear to be related to other known (1,3)-a-GTs, such as the human blood group A (1,3)-a-N-acetylgalactosaminyltransferase, assigned to CAZy GT-family-6, or the a-(1,6)-or the (1,2)-b-D-xylosyltransferases involved in the xyloglucan and N-glycan biosynthesis and assigned to CAZy GT-family-34 and -61, respectively. RGXT1 and RGXT2 were thus used to seed CAZy GT-family-77, which presently comprises 34 accessions: 18 from Arabidopsis, 15 from O. sativa, and a single Dictyostelium discoideum (1,3)-a-D-galactosyltransferase (Ketcham et al, 2004), which also catalyzes the formation of an a-(1,3)-linkage to fucose. Classification of a GT to a particular CAZy family is very strong evidence for catalytic mechanism, retaining or inverting, and the type of linkage formed generally also correlates well with family membership.…”

The Skp1 Prolyl Hydroxylase from Dictyostelium Is Related to the Hypoxia-inducible Factor-α Class of Animal Prolyl 4-Hydroxylases

“…Frozen cell pellets were resuspended on ice in E. coli lysis buffer (0.1 M Tris-HCl (pH 8.2), 1 mg/ml lysozyme, 5 mM benzamidine, 0.5 g/ml pepstatin A, 5 g/ml aprotinin, 5 g/ml leupeptin, and 0.5 mM phenylmethylsulfonyl fluoride) at 1 ml/25 ml of the original culture volume, lysed in a French pressure cell (15,000 p.s.i. ), and centrifuged at 100,000 ϫ g for 1 h. The supernatant was purified by successive chromatography over a DEAE-Sepharose fast flow anion exchange column and a phenyl-Sepharose fast flow (Hi-Sub) column (both from Amersham Biosciences) (14). The pool of Skp1 that eluted in the ascending ethylene glycol gradient was further purified and concentrated over a Source 15Q anion exchange column (Amersham Biosciences) eluted with an ascending gradient of NaCl in 25 mM NH 4 Ac (pH 7.5), 5 mM MgCl 2 , 15% (v/v) glycerol, 1 mM dithiothreitol, and protease inhibitors (10 g/ml leupeptin, 10 g/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride).…”

“…The catalytic domains of the diglycosyltransferase are each related to separate families of cytoplasmically associated glycosyltransferase domains of bacteria and eukaryotes (12). The next sugar, ␣-3-linked Gal, is attached by a separate soluble enzyme (14). The final glycan formed consists of a type 1 blood group H structure with peripheral ␣-linked Gal modifications: Gal␣1,Gal␣1,-3Fuc␣1,2Gal␤1,3GlcNAc␣1-.…”

Glycosylation of Skp1 Promotes Formation of Skp1–Cullin-1–F-box Protein Complexes in Dictyostelium

“…Gel Filtration of Cytosolic Extracts-Vegetative (growth-stage) cells were harvested as described above; resuspended in 50 mM Tris-HCl (pH 7.4), 0.25 M sucrose, and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 10 g/ml aprotinin, added just before use); and lysed by means of forced filtration through a 5-m pore diameter filter as described previously (17). A cytosolic extract was prepared via ultracentrifugation (100,000 ϫ g for 60 min) at 4°C and chromatographed on a Superose 12 gel filtration column at 22°C pre-equilibrated in 50 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1 mM DTT, and 10% glycerol.…”