phytanyl phosphate6c). Thus, polar groups appear to be tolerated
at the w-position. The soluble biotinylated compounds 4a and
4b were less good with relative activities of 17 and 11%, but
were nevertheless clearly recognised and mannosylated by the
enzyme.
The Dol-P-Man synthase was then tested for activity against
the solid-supported substrates 5a,b and 6 using the same assays
as before, measuring incorporation of radioactivity from GDP-
14C-mannose into lipid.6g Incorporation of mannose into lipid
could be monitored either by directly counting radioactivity on
solid support or after cleavage off the support with biotin (for 5a
and 5b) or mercaptoethanol (for 6). All solid-supported
compounds 5a, 5b and 6 appeared to be substrates for the Dol-
P-Man synthase, since a significant amount of the radiolabelled
starting material was converted to product (1190 cpm for 5a and
970 cpm for 5b; 3120 cpm for 6; blank (no lipid) gave 65
cpm).
A more detailed analysis was carried out on the incubation
products of 6 using LC-MS. After mercaptoethanol cleavage of
the incubation products, 13 and 14 are clearly detectable by LC-
MS next to starting materials 15 and 16 (Fig. 1).
These results demonstrate that bifunctional lipids 3U6 are
substrates for the Dol-P-Man synthase present in the crude
microsomes of yeast. Enzymatic mannosylation of phosphate
lipids should be a useful route to Dol-P-Man substrate
analogues, since the b-mannosyl phosphate linkage is partic-
ularly difficult to synthesise.10 In addition, we have shown for
the first time that a dolichol dependent glycosyltransferase can
recognise substrates on solid support. These findings open up
potential applications in enzyme-catalysed solid phase synthe-
sis of N-glycans.
Finally, it is interesting to note a practical advantage of solid
phase enzymatic synthesis: the product isolation consists simply
of washing the resin and can thus tolerate the use of crude
enzyme preparations such as the microsomal fractions in the
present study. Since purification of glycosyltransferases, in
particular of the membrane associated dolichol dependent
transferase is difficult, solid phase synthesis is attractive.
We thank the Deutsche Forschungsgemeinschaft (DFG) for a
research fellowship to I. S., the EC (ERASMUS) for support to
A. Z., the BBSRC for grant support and the Wellcome Trust for
support of the Edinburgh Protein Interaction Centre (EPIC).
Notes and references
‡
Yeast microsomes were prepared as described before.6g
A typical enzymatic assay: acceptor lipids 2, 3, 4a and 4b (100 mM) were
incubated, each with crude microsomes (1U2 mg), GDP-mannose (20 mM),
GDP-[U-14C]mannose (0.1 mCi mL21) and buffer (50 mM Tris-HCl, 5 mM
MgCl2, 10 mM mercaptoethanol, 0.5% Triton-X-100 (v/v), pH 7.5, 1 mL)
at 37 °C for 1 h. Aliquots (100 mL) were removed and added to 100 mL of
CHCl3UMeOH (1+1, v/v). The aqueous phase was removed after centrifuga-
tion. The organic phase was washed twice with water and incorporation of
radioactivity into organic soluble material was measured by scintillation
counting.
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