Substrate Specificity of the Dolichol Phosphate Mannose: Glucosaminyl Phosphatidylinositol α1-4-Mannosyltranferase of the Glycosylphosphatidylinositol Biosynthetic Pathway of African Trypanosomes

Abstract

The biosynthesis of glycosylphosphatidylinositol (GPI) precursors in Trypanosoma brucei involves the D-mannosylation of D-GlcN␣1-6-D-myo-inositol-1-PO 4 -sn-1,2-diacylglycerol (GlcN-PI). An assay for the first mannosyltransferase of the pathway, Dol-P-Man:GlcN-PI ␣1-4-mannosyltransferase, is described. Analysis of the acceptor specificity revealed (a) that the enzyme requires the myo-inositol residue of the GlcN-PI substrate have the D configuration; (b) that the enzyme requires the presence of the NH 2 group of the D-GlcN residue; (c) that GlcNAc-PI is more efficiently presented to the enzyme than GlcN-PI, suggesting a degree of substrate channelling via the preceding GlcNAc-PI de-N-acetylase enzyme; (d) that the fatty acid and phosphoglycerol components of the phosphatidyl moiety are important for enhancing substrate presentation and substrate recognition, respectively; and (e) that D-GlcN␣1-6-D-myoinositol is the minimum structure that can support detectable acceptor activity. Analysis of the donor specificity revealed that short chain (C 5 and C 15 ) analogues of dolichol phosphate can act as substrates for the trypanosomal dolichol-phosphomannose synthetase, whereas the corresponding mannopyranosides cannot act as donors for the Dol-P-Man:GlcN-PI ␣1-4-mannosyltransferase.

Substrate specificity of the N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol membrane anchor biosynthesis in African trypanosomes and human cells

“…This is presumably due to shorter acyl chains being attached to the glycerol backbone in synthetic GlcN-(sn-1,2-diplamitoyl)PI compared with those of endogenous intermediates [21]. As noted previously, the addition of GlcNAc-PI produces substantially more mannosylated GPI intermediates than does the addition of GlcN-PI (compare Figure 2, lanes 3 and 4), suggesting that GlcN-PI is best presented to the α-mannosyltransferases via the de-N-acetylase [11]. The compounds GlcNPr-PI, GlcNBu-PI, GlcNiBu-PI and GlcNPen-PI were also able to undergo de-N-acylation and subsequent mannosylation ( Figure 2, lanes 5-8), whereas GlcNHex-PI showed no detectable de-Nacylation\mannosylation activity (Figure 2, lane 9).…”

“…It is worth noting that the trypanosome cell-free system processes GlcNAc-PI roughly 6 times more efficiently than GlcN-PI ( [11] and Figure 2, compare lanes 3 and 4) whereas the HeLa cell-free system processes GlcN-PI roughly 1n5 times more efficiently than GlcNAc-PI ( Figure 6, compare lanes 2 and 4). This observation may be significant since it has been suggested that the enhanced processing of GlcNAc-PI over GlcN-PI indicates a degree of substrate channelling between the trypanosome de-N-acetylase and the first α-mannosyltransferase [11].…”

“…This observation may be significant since it has been suggested that the enhanced processing of GlcNAc-PI over GlcN-PI indicates a degree of substrate channelling between the trypanosome de-N-acetylase and the first α-mannosyltransferase [11]. Such substrate channelling between these two enzymes is not evident in the HeLa cell-free system, possibly because acylation of the inositol ring of the GlcN-PI intermediate is a prerequisite for mannosylation in mammalian cell-free systems [27].…”

“…GlcNAc-[$H]PI, the natural substrate, underwent " 36 % turnover to GlcN-PI under these conditions (Figure 1, lanes 3 and 4), whereas the turnover of GlcNPr- The specificity of the GlcNAc-PI de-N-acetylase for the N-acyl group was also investigated using an indirect assay based on the ability of chemically synthesized (non-radiolabelled) GlcNR-PI substrates to undergo mannosylation by the trypanosome cellfree system. Previous studies showed that de-N-acetylation of exogenous GlcNAc-PI precedes the addition of the three αMan residues in the GPI biosynthetic pathway [11] so that mannosylation of exogenous substrates can be used as an indirect (but highly sensitive) measure of GlcNR-PI de-N-acylation.…”

“…A Chinese hamster ovary cell mutant that is deficient in GlcNAc-PI de-N-acetylase activity has been described recently [10] but the defective gene has yet to be isolated. De-N-acetylation is a prerequisite for the mannosylation of GlcN-PI to form later GPI intermediates [11] and it has been suggested that this step is regulated in mammalian cells by GTP [9].…”