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Phosphomannomutase catalyses the production of mannose-1 -phosphate from mannose- 6-phosphate in the pathway leading to the production of GDP-Man (Figure 1.13). A deficiency of phosphomannomutase would in theory lead to a decrease in the amount of m annose-1 -phosphate, GDP-Man and dolichol-P-Man. Mannose-6-phosphate would probably not accumulate as there are other pathways available for its utilisation. The dolichol-linked oligosaccharide precursor contains nine mannose residues all of which are obtained from either GDP-Man or dolichol-P-Man. If either or both of these substrates are decreased, then a slower rate of synthesis of the precursor would be expected. It has been shown by Panneerselvam and Freeze (1996a) that there is reduced GDP-Man, but not dolichol-P-Man, and the rate of synthesis of the lipid-linked oligosaccharide

precursor is indeed decreased in CDGS type 1 (PMM deficient) fibroblasts. The lipid- linked oligosaccharide precursor is the major substrate for oligosaccharyltransferase in the protein N-glycosylation reaction. Hence when there is a limited supply of this substrate some glycosylation sequons will not become glycosylated. This phenomenon would lead to the under-glycosylation of glycoproteins as observed in CDGS type 1 patients.

The genetic locus o f CDGS type 1A (PMM-deficient) has been assigned to chromosome 16pl3 (Martinsson et a l, 1994). The genetic locus of PMM-normal CDGS type 1 is elsewhere (Matthijs et a l, 1996,1997a). Eleven missense mutations have been found in PMM2 in 16 PMM-deficient CDGS type 1 patients (Matthijs et a l, 1997b). As yet there are no reports of expression of either the wild type human PMM2 or mutant PMM2 with which to confirm that these mutations are actually disease causing mutations.

1.5.6.3 Phosphoglucomutase (EC 5.4.2.2)

Phosphoglucomutase is the enzyme which forms the link between catabolism of glycogen with the glycolytic degradation pathway. In the reverse direction it contributes to the formation o f UDP-Glc for the biosynthesis of glycoproteins (Boles, 1994). PGM exists as at least five isoenzymes; PG M l, PGM2, PGM3, PGM4, and PGM5, which are encoded by five distinct genes on chromosomes 1,4, 6, unassigned and 9 respectively (Drago et a l , 1991 ; Edwards et a l, 1995). They are thought to have arisen by

duplication o f a common ancestral gene. The proteins are all single monomers having a molecular weight o f around 60 kDa and they have diverged to have different substrate specificity by point mutations (Whitehouse et a l, 1992). PGM l is immunologically distinct from PGM2 and PGM3 as antibodies against PGM l do not cross-react with PGM2 or PGM3, however they do cross react with PGM4 (Drago et a l, 1991). PG M l activity dominates in most cells, except for red blood cells were PGM2 is highly active and the lactating mammary gland where PGM4 is the most active (Putt et a l, 1993).

The complete sequence of human PGM l is known (Whitehouse et a l, 1992) and the crystal structure of the enzyme from rabbit muscle has been identified (Lin et a l, 1986; Dai et a l , 1992). Rabbit muscle PGM l has four domains, all of which contribute to the

active site cleft. Activation of the enzyme occurs via phosphorylation o f serine 116, which lies in the bottom of the cavity between the protein domains, and the phosphates associate with arginines 426, 514 and 292. The bivalent metal ion activator region is also found in the cavity, and so the enzyme has a physiological requirement for Mg^"^. The reaction is thought to take place within the confines of the cavity (Lin et al., 1986, Dai et a l, 1992). PG M l consists of primary and secondary isoenzymes and is highly

polymorphic and hence has been widely used in forensic science (Whitehouse et a l , 1992). PG M l can be distinguished as two alloenzymes by starch gel electrophoresis, they are designated PGM1*1 and PGM 1*2. PGM 1*2 has a greater anodal

electrophoretic mobility (March et a l, 1993). Isoelectric focusing of PG M l further divides these alloenzymes into acidic enzymes (+) and basic enzymes (-). The molecular basis o f the different isoenzymes of PGMl seen on starch gel electrophoresis has been attributed to two point mutations. A change from Arg to Cys at residue 220 causes the PG M l* 1/2 polymorphism, whereas a change from Tyr to His at residue 419 causes the PG M l +/- polymorphism. It is thought that these polymorphisms have arisen by

intragenic recombinations (Whitehouse et a l, 1992, March et a l, 1993; Putt et a l , 1993, Takahashi and Neel, 1993).

The phosphoglucomutases have broad specificity and can act on many sugar phosphates including mannose-l-phosphate. PGM2 has the highest phosphomannomutase activity (Putt et a l, 1993). Indeed, in mammals it was assumed until recently that

phosphoglucomutase did interconvert mannose-1 -phosphate and mannose-6-phosphate in the glycosylation pathway (Ray and Peck, 1972). Phosphomannomutase can also convert glucose-6-phosphate to glucose-1-phosphate as PGM double mutants still have some residual activity (Whitehouse et a l, 1992). PGM and PMM are however distinct enzymes as PGM genes have not been found on chromosome 16 or chromosome 22. However as PGM2 is known to have considerable phosphomannomutase activity and there is residual activity of phosphomannomutase in CDGS type 1 patients, the expression of the PGM isoenzymes in these patients requires further investigation.

Phosphoglucomutase is unusual in that it is a cytoplasmic protein which contains an oligosaccharide containing mannose, glucose and phosphate. It is believed that this post-

translational modification is important for modulating the function of PGM just as phosphorylation modulates other enzymes (Marchase, 1993).