263
Ann. Hum.Genet. (1991), 55, 263-271 Printed in Great Britain
Antigenic analysis of the major human phosphoglucomutase isozymes : PGM1, PGM2, PGM3 and PGM4 G. A. DRAGO, D. A. HOPKINSON, S. A. WESTWOOD' AND D. B. WHITEHOUSE MRC Human Biochemical Genetics Unit, The Galton Laboratory, University College London, Wolfson House, 4 Stephenson Way, London NWl 2HE, UK
SUMMARY
The cross-reactivity of human phosphoglucomutase isozymes (PGMl, PGM2, PGMS and PGM4) has been investigated using anti-rabbit muscle PGM polyclonal antibodies. Significant differepces were revealed: an IgG fraction of the antiserum reacted with the primary and secondary PGMl isozymes of all the common phenotypes. However, there was no reaction with the PGM2 or PGM3 isozymes ; thus these latter isozymes share no major antigenic determinants with human or rabbit PGMl and are therefore structurally distinct. I n contrast, the PGM isozymes of human milk attributed to a fourth locus, PGM4, showed similar cross-reactivity as PGMl suggesting close structural similarity. The IgG was also employed as a'reagent to remove PGMl from haemolysates so as to allow the unambiguous assessment of the PGMS isozyme patterns by isoelectric focusing. However, no proven genetic variation was encountered in a sample of 32 individuals. INTRODUCTION
Phosphoglucomutase (EC 5.4.2.2) is a soluble intracellular enzyme that catalyses the interconversion of glucose-1-phosphate and glucose-6-phosphate ; it has a major function in glycogen metabolism (Ray & Peck, 1972). Following starch gel electrophoresis of a wide range of human tissues, a characteristic series of phosphoglucomutase (PGM) isozymes can be detected that are encoded by three independent autosomal gene loci designated PGM1, PGM2 and PGMS (McAlpine et al. 1990). On the basis of biochemical and physical similarities, the isozymes encoded by the three structural loci are thought t o constitute an ancient gene family that evolved by duplication of a common ancestral gene. Whilst there has been subsequent divergence as a result of point mutations leading to differences in substrate handling, all three enzymes are single-chain monomers of around MW 60000 (McAlpine et al. 1970a; Whitehouse et al. 1991). In addition, there is a unique set of isozymes in human milk thought to be the product of a fourth gene locus, designated PGM4 (Cantu & Ibarra, 1982). The PGMl locus is highly polymorphic and has become a well established biochemical marker in human genetics (Spencer et al. 1964; Carter et al. 1979; Sensabaugh & Crim, 1982). During a preliminary immunological investigation of the PGMl isozyme polymorphism, an anti-rabbit muscle PGM antiserum was produced in a male sheep that cross-reacts with human PGMl (Whitehouse et al. 1989). When used as a probe to identify PGMl on immunoblots of isoelectric focusing gels, this reagent appeared t o be monospecific and detected all of the focused CRSE, Home Office Forensic Service, Aldermaston, Reading, Berkshire, RG7 4PN. 19
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G. A. DRAGO AND
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components of the common PGMl phenotypes without interference from any other immunoreactive bands. Recently the antiserum was employed to identify a cDNA sequence encoding the entire rabbit PGMl protein by screening a h g t l l expression library constructed using rabbit muscle mRNA. This has led directly to the isolation and characterization of a full human PGM cDNA sequence (Whitehouse et al. 1991). I n this report we provide further information about the crossreacting properties of this antirabbit PGMl serum. Using electrophoresis and enzyme activity staining to analyse human haemolysates, placental and milk extracts following treatment with an anti-rabbit PGMl IgG fraction, we have determined the extent of the cross-reactivity with all of the major isozymes of the human PGM gene family.
MATERIALS AND METHODS
Sump1es
Human haemolysates, placentae and breast milks were investigated. Haemolysates were prepared by freezing and thawing packed washed red cells. Aqueous placental extracts were prepared by homogenizing 1 g of tissue in 2 ml distilled water, followed by centrifugation a t 1 O O O O g for 15 min a t 4 "C. The clear supernatants were used for immunoreactivity studies. Milk samples were prepared by centrifugation a t 1000 g for 15 min. The lower (non-fat)layer was collected for study. Antibodies
Polyclonal antibodies to purified rabbit muscle phosphoglucomutase were raised in a sheep using a standard protocol (Whitehouse et al. 1989). An IgG fraction of the antiserum was prepared by affinity chromatography using a 5-0 ml Sepharose-protein G column as directed by the manufacturer (Pharmacia). Antibody treatments
Various aqueous dilutions of an IgG fraction of the antiserum were mixed with neat haemolysate, placental extract or defatted milk and incubated a t 4 "C for 4 h. The total reaction volume used was between 100 and 200 ,ul according to the experiment. This material was used for analysis without further modification. I n all experiments the untreated control samples were adjusted to the same dilution as the treatments with distilled water, Electrophoresis
Starch gel electrophoresis was conducted using a Tris-EDTA-maleate buffer system (Harris & Hopkinson, 1976). Equilibrium polyacrylamide gel isoelectric focusing was performed using
a pH 5-7 gradient (Whitehouse et al. 1989). The zones of PGM activity were detected using a specific reaction mixture applied to the gel in an agar overlay (Harris & Hopkinson, 1976).
Antigenic analysis of PGM
265
4PGM2 PGMl
-Hb
1
2
3
4
5
Fig. 1. Starch gel electrophoresis showing immunoprecipitation of PGMl in a haemolysate (phenotype PGMl 1) with various dilutions of IgG as follows: (2) 1 in 2 ; (3) 1 in 3 ; (4)1 in 4;(1) and ( 5 ) are untreated controls. The positions of the normal P G M l and PGM2 isozymes are indicated.
RESULTS
PGMl The PGM isozyme patterns of human haemolysates following incubation with various dilutions of an anti-rabbit PGMl IgG fraction were analysed by starch gel electrophoresis and enzyme activity staining. It can be seen that PGMl was completely inactivated by reaction with an IgG dilution of 1 in 2 (Fig. 1 ) . It seems most likely that inactivation was due to immunoprecipitation, since the IgG precipitated human skeletal muscle PGM in Ouchterlony double diffusion plates. One weakly staining precipitin line was produced which fused with the major precipitin line produced by interaction of the IgG with purified rabbit muscle PGM, without forming a spur in either direction. At higher IgG dilutions ( 1 in 3 and 1 in 4,Fig. l ) , a single component of PGM activity was observed that migrated more slowly than the PGMl isozymes in the untreated controls. This component was of consistently lower activity than the control PGMl and was probably due to the presence of unprecipitated PGMl enzyme-antibody complexes. To investigate the reaction of the antibodies with the PGMl isozymes by immunobinding as opposed to liquid immunoprecipitation, several 0 5 ml protein G Sepharose columns were prepared in modified 1.5 ml microcentrifuge tubes. 250 ,ul of neat anti-rabbit PGMl antiserum was loaded on to each column which was then washed with 0.05 M-Na phosphate buffer (pH 7-0) to remove unbound material. The resulting protein G-anti-rabbit PGMl IgG column was employed for immunoaffinity chromatography. 250 ,ul of haemolysate was applied to the column and the unbound fraction collected by centrifugation a t 800 g for 5 min and analysed 19 2
G. A. DRAGO AND
266
OTHERS
+ PGM2 PGMl
-Hb
+ PGMl phenotype
1
+
3
2-1
+
1
-
+ 2-1
Fig. 2. Starch gel electrophoresis showing PGM isozyme patterns from four haemolysates before ( - ) and following ( + ) immunoaffinity chromatography. Experiments with two PGMl phenotypes (1 and 2-1) are illustrated.
using starch gel electrophoresis. Figure 2 shows that the immunoaffinity columns removed all of the PGMl activity in the samples. There was no trace of the slowly migrating isozymes(s) seen in the previous experiment with the higher IgG dilutions (Fig. 1). Seventeen haemolysates representing four common PGMl phenotypes (1+, l+l-,2-1+, 2+l+) were tested for crossreaction by incubating 80 p1 of haemolysate with 20 pl of undiluted IgG fraction (1 in 5) and analysed by isoelectric focusing, gradient pH 5-7 (Fig. 3). In each case the PGMl isozyme activity was completely abolished. Thus it was concluded that there were no major differences in the antigenic properties of the products of the four common alleles ( l + ,1-, 2+, 2-) found in haemolysates. This is in keeping with immunoblotting studies where the IgG was shown to react with the PGMl isozymes of the common phenotypes immobilized on nitrocellulose filters (Whitehouse et al. 1989).
PGM2 It is evident from the liquid immunoprecipitation experiments that the human PGM2 isozymes in the haemolysates were completely nonreactive with the anti-rabbit PGMl antibodies, even a t the lowest dilution of the IgG fraction employed (Figs 1 and 3). Furthermore, the PGM2 isozymes passed through the immunoaffinity columns without marked alteration (Fig. 2). This suggests that the PGMl and PGM2 proteins are antigenically distinct.
Antigenic analysis of PGM
267
acidic
basic
+ PGMl phenotype:
-
2+1+
+ - + - + - + 2+1+ 2+1+
1+1-
-
2-1+
+ - + - + 1+1’
2-1+
2+1+
Fig. 3. Isoelectric focusing analysis of erythrocyte PGM in a pH 5-7 gradient before ( - ) and following ( + ) treatment with a 1 in 5 dilution of IgG. Three common PGMl subtypes are illustrated.
basic
*
c
Fig. 4. Isoelectric focusing in a pH 5-7 gradient showing erythrocyte PGM2 isozyme patterns from unrelated individuals following removal of PGMl activity by immunoaffinity chromatography. The possible variant phenotype is indicated (*). An untreated control sample (C), phenotype PGM 1+1-, is included for comparison.
G. A. DRAGO AND
268
OTHERS
+
PGM3 PGM2 PGMl
1
2
3
Fig. 5. Starch gel electrophoresis showing the PGM isozymes in a placental extract following treatment with various dilutions of IgG as follows: (2) 1 in 2; (3) 1 in 3 ; (1) is an untreated control. The PGM2 and PGM3 isozymes are unaffected by the treatment with IgG. Prolonged incubation, leading to overstaining of PGMl and PGM2, was necessary to visualize the PGM3 isozymes.
The capacity of the IgG to remove all of the PGMl activity from the pattern provided a novel opportunity of conducting an unambiguous assessment of the PGM2 pattern on high resolution isoelectric focusing gels. This analysis was performed using a small population sample (n = 32) to determine if hitherto undiscovered common genetic variation a t the PGM2 locus could be detected. As anticipated, treatment of the samples either with a 1 in 5 dilution of IgG or by using immunoaffinity chromatography, resulted in the complete removal of the PGMl activity from the samples. Following isoelectric focusing in gradient p H 5-7 gels and activity staining, a complex series of PGM2 isozymes was resolved (Figs 4 and 3). No proven genetically determined person-to-person variation was found in this small sample. A single sample, from a north European individual, was found to have a variant PGM2 pattern which could have been heterozygous. Unfortunately, it was not possible to obtain a repeat blood sample from this person or to conduct a family study. Nonetheless, the approach of utilizing the IgG as a reagent to remove ‘contaminating’ PGMl components is likely to be a useful adjunct in further isoelectric focusing studies of PGM2 variation.
PGM3 The immunoreactivity of the human PGM3 isozymes was investigated using placental extracts as a source of the enzyme. Following incubation of extracts with a 1 in 3 dilution of the IgG fraction and starch gel electrophoresis, it can be seen that there was no crossreaction with
Antigenic analysis of PGM
269
+
1
2
3
4
5
6
7
8
9
Fig. 6. Starch gel electrophoresis showing PGM4 isozymes in untreated milk samples from two mothers (AG and CL) and complete removal of PGM4 activity by immunoprecipitation using various IgG dilutions: (3) AG untreated; (4) AG 1 in 5 ; ( 5 ) AG 1 in 10; (6) AG 1 in 20; ( 7 ) CL untreated; (8) CL 1 in 5 ; (9) CL 1 in 10. A haemolysate, before ( 1 ) and after (2) IgG treatment, is also illustrated.
the PGM3 isozymes (Fig. 5 ) . Complete removal of normal PGMl activity from the placental extract was achieved with all antibody dilutions and, as in the previous experiments, the PGM2 activity was undiminished. Interestingly, the amount of slowly migrating enzyme-antibody complexed material was more abundant in these placental extracts than in the haemolysates. This reflects the relatively high level of PGMl activity in placenta. It can be seen that the activity of the PGM-antibody complexes increased as the concentration of the reagent antibodies decreased.
PGM4 in milk To investigate PGM4, defatted milk sample from two mothers were incubated with various dilutions of the IgG fraction and analysed by starch gel electrophoresis and specific activity staining (Fig. 6). I n both cases the PGM4 isozyme activity was completely removed, even a t an IgG dilution of 1 in 20. This was about four times less anti-rabbit PGMl IgG than was required to remove all of the PGMl activity from haemolysates. At higher dilutions of IgG traces of PGM4 isozyme activity with normal electrophoretic mobility were observed. PGM4 isozyme patterns were also investigated by isoelectric focusing. Eighteen samples were examined before and after treatment with the IgG fraction. Considerable variation in the PGM4 isozyme pattern was identified but in every case all of the milk PGM activity was removed by treatment with the IgG (Fig. 7).
G. A. DRAGO AND
270
OTHERS
acidic
basic
-
+
+
1
+ - + - +
-
+
I
+
+
I +
I
+
I
rbc
rbc
Fig. 7 . Isoelectric focusing in a pH 5-7 gradient of PGM4 in eight milk samples before ( - ) and after ( ) treatment with a 1 in 5 dilution of IgG and two haemolysate controls (rbc). PGM4 isozyme variation is evident in the untreated milk samples.
+
DISCUSSION
Phosphoglucomutase is encoded by multiple gene loci that are thought to constitute a structurally related gene'family . We have employed polyclonal antibodies raised against rabbit muscle PGMl to investigate the antigenic relationships of human PGM1, PGM2, PGMS and PGM4. Of the well-characterized isozymes encoded by PGMl, PGMB and PGMS, only PGMl was found to be immunoreactive. Furthermore, the primary and secondary isozymes of all of the variant PGMl phenotypes examined reacted equally well with the antibodies. Thus PGMl is not immunologically related to PGM2 or PGM3. This conclusion suggests that if the three loci arose by duplication of a common ancestor gene, sufficient mutational differences have occurred to render the three sets of isozymes antigenically distinct. This level of structural divergence is in keeping with the divergent biochemical properties of the PGM isozymes (Quick et al. 1974; Fisher & Harris, 1972; McAlpine et al. 1970b, c ) . Although it is feasible that the heterospecific anti-rabbit antibodies may recognize only a small number of the potential antigenic determinants on human PGMl, it has been shown that there is 97 'YO homology between the rabbit and human PGMl derived amino-acid sequences (Whitehouse et al. 1991).Therefore i t is reasonable to assume that the majority of the PGMl antigenic determinants will be conserved between the two species. An independent study using anti-human polyclonal reagents has also produced strong evidence that the human erythrocyte PGMl and PGMB isozymes are not immunologically related (Piatti et al. 1990). The origin and properties of the unique human PGM4 isozymes are less well understood. Analysis of a putative genetic polymorphism of PGM4 suggests it is independent of the PGMl locus (Cantu & Ibarra, 1982), although family studies were not possible and heritability of the isozyme patterns could not be proved. PGM4 isozymes are found only in milk, suggesting that their regulation differs considerably from PGMl ; furthermore, milk appears t o be completely devoid of activity associated with the expression of PGM1, PGMS and PGMS. The antigenic
Antigenic analysis of PGM
27 1
similarity of the PGMl and PGM4 isozymes, taken together with their shared catalytic properties, would be in keeping with a recent gene duplication where considerable sequence homology has been preserved. However, in the absence of unambiguous inherited variation, the data are also consistent with an alternative origin of PGM4 from post-transcriptional or posttranslational processing of PGMl gene products. The method we have employed to determine the immunoreactivities of the PGM isozymes using electrophoresis is rapid, simple to perform and provides a broad qualitative assessment of antigenic properties. For instance the reactivities of both primary and secondary isozymes as well as a range of phenotypes of PGM can be assessed in a single experiment. However, the determination of features such as degree of antibody binding would be better accomplished using a more quantitative technique such as ELISA or enzyme assay. It is expected that the immunological approach will continue to provide useful information on structural features differentiating the isozymes encoded by the members of the human PGM gene family. It will be particularly interesting to investigate the reactions of monoclonal antibodies to determine if individual epitopes associated with linear regions of the protein vital for catalytic activity have been conserved by all the members of the gene family. G. A. Drago was supported by a grant from the Home Office.
REFERENCES
CANTU,J. M. & IBARRA, B. (1982).Phosphoglucomutase : evidence for a new locus expressed in milk. Science 216, 639-640. CARTER,N. D., WEST, C. M., EMES, E., PARKIN, B. & MARSHALL,W. H. (1979). Phosphoglucomutase polymorphism detected by isoelectric focusing : gene frequencies, evolution and linkage. Ann. Hum. Biol. 6, 22 1-230. FISHER, R. A. & HARRIS,H. (1972). ‘Secondary’ isozymes derived from the three PGM loci. Ann. Hum. Genet. 36, 69-77. HARRIS,H. & HOPKINSON, D. A. (1976). Handbook of Enzyme Electrophoresis in Human Genetics, 2.7.5.1, 1. Amsterdam : North-Holland Publishing Co. MCALPINE,P. J., STRANC, L. C., BOUCHEIX, C. & SHOWS, T. B. (1990).The 1990 catalog of mapped genes and report of the nomenclature committee. Cytogenet. Cell Genet. 5 5 , 5-76. MCALPINE,P. J . , HOPKINSON, D. A. & HARRIS,H. (1970a). Molecular size estimates of human phosphoglucomutase isozymes by gel filtration chromatography. Ann. Hum. Genet. 34, 177-185. MCALPINE,P. J., HOPKINSON, D. A. & HARRIS, H. (1970b).Thermostability studies on the isozymes of human phosphoglucomutase. Ann. Hum. Genet. 34, 61-71, MCALPINE, P. J., HOPKINSON, D. A. & HARRIS,H. ( 1 9 7 0 ~ )The . relative activities attributable to the three phosphoglucomutase loci (PGM,, PGM, and PGM,) in human tissues. Ann. Hum. Genet. 34, 169-175. PIATTI,E., ACCORSI, A,, PIACENTINI, M. P. & FAZI,A. (1990). Immunological studies on erythrocyte phosphoglucomutase isozymes. Biochem. Int. 21, 155-163. QUICK,C. B., FISHER, R. A. & HARRIS,H. (1974). A kinetic study of the isozymes determined by the three phosphoglucomutase loci PGM,,PGM, and PGM,.Europ. J. Biochem. 42, 511-517. RAY,W. J., JR& PECK, E. J., J R . (1972).Phosphomutases. I n The Enzymes, vol. 6, 3rd edn (ed. P. D. Boyer), pp. 407-477. New York, London: Academic Press. QENSABAUGH, G. F. & CRIM,D. (1982).Biochemical markers of individuality. I n Forensic Science Handbook (ed. R. Saferstein), pp. 338415. New Jersey : Prentice-Hall Inc. N., HOPKINSON, D. A. & HARRIS,H. (1964). Phosphoglucomutase polymorphism in man. Nature 204, SPENCER, 742-745. WHITEHOUSE,D. B., DRAGO,G. A,, HOPKINSON,D. A,, WESTWOOD,S. A. & WERRETT,D. J. (1989). Immunological detection of human phosphoglucomutase (PGM1) subtypes. Forensic Sci. Int. 41, 25-34. WHITEHOUSE, D. B., PUTT, W., LOVEGROVE, J . U., MORRISON, K., HOLLYOAKE, M., Fox, M., HOPKINSON, D. A. & EDWARDS, Y. H. (1991).Phosphoglucomutase 1 (PGMl); complete human and rabbit mRNA sequences and direct mapping of this highly polymorphic marker on human chromosome 1. Proc. Natl. Acad. Sci.USA (in press).
Ann. Hum.Genet. (1991), 55, 263-271 Printed in Great Britain
Antigenic analysis of the major human phosphoglucomutase isozymes : PGM1, PGM2, PGM3 and PGM4 G. A. DRAGO, D. A. HOPKINSON, S. A. WESTWOOD' AND D. B. WHITEHOUSE MRC Human Biochemical Genetics Unit, The Galton Laboratory, University College London, Wolfson House, 4 Stephenson Way, London NWl 2HE, UK
SUMMARY
The cross-reactivity of human phosphoglucomutase isozymes (PGMl, PGM2, PGMS and PGM4) has been investigated using anti-rabbit muscle PGM polyclonal antibodies. Significant differepces were revealed: an IgG fraction of the antiserum reacted with the primary and secondary PGMl isozymes of all the common phenotypes. However, there was no reaction with the PGM2 or PGM3 isozymes ; thus these latter isozymes share no major antigenic determinants with human or rabbit PGMl and are therefore structurally distinct. I n contrast, the PGM isozymes of human milk attributed to a fourth locus, PGM4, showed similar cross-reactivity as PGMl suggesting close structural similarity. The IgG was also employed as a'reagent to remove PGMl from haemolysates so as to allow the unambiguous assessment of the PGMS isozyme patterns by isoelectric focusing. However, no proven genetic variation was encountered in a sample of 32 individuals. INTRODUCTION
Phosphoglucomutase (EC 5.4.2.2) is a soluble intracellular enzyme that catalyses the interconversion of glucose-1-phosphate and glucose-6-phosphate ; it has a major function in glycogen metabolism (Ray & Peck, 1972). Following starch gel electrophoresis of a wide range of human tissues, a characteristic series of phosphoglucomutase (PGM) isozymes can be detected that are encoded by three independent autosomal gene loci designated PGM1, PGM2 and PGMS (McAlpine et al. 1990). On the basis of biochemical and physical similarities, the isozymes encoded by the three structural loci are thought t o constitute an ancient gene family that evolved by duplication of a common ancestral gene. Whilst there has been subsequent divergence as a result of point mutations leading to differences in substrate handling, all three enzymes are single-chain monomers of around MW 60000 (McAlpine et al. 1970a; Whitehouse et al. 1991). In addition, there is a unique set of isozymes in human milk thought to be the product of a fourth gene locus, designated PGM4 (Cantu & Ibarra, 1982). The PGMl locus is highly polymorphic and has become a well established biochemical marker in human genetics (Spencer et al. 1964; Carter et al. 1979; Sensabaugh & Crim, 1982). During a preliminary immunological investigation of the PGMl isozyme polymorphism, an anti-rabbit muscle PGM antiserum was produced in a male sheep that cross-reacts with human PGMl (Whitehouse et al. 1989). When used as a probe to identify PGMl on immunoblots of isoelectric focusing gels, this reagent appeared t o be monospecific and detected all of the focused CRSE, Home Office Forensic Service, Aldermaston, Reading, Berkshire, RG7 4PN. 19
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OTHERS
components of the common PGMl phenotypes without interference from any other immunoreactive bands. Recently the antiserum was employed to identify a cDNA sequence encoding the entire rabbit PGMl protein by screening a h g t l l expression library constructed using rabbit muscle mRNA. This has led directly to the isolation and characterization of a full human PGM cDNA sequence (Whitehouse et al. 1991). I n this report we provide further information about the crossreacting properties of this antirabbit PGMl serum. Using electrophoresis and enzyme activity staining to analyse human haemolysates, placental and milk extracts following treatment with an anti-rabbit PGMl IgG fraction, we have determined the extent of the cross-reactivity with all of the major isozymes of the human PGM gene family.
MATERIALS AND METHODS
Sump1es
Human haemolysates, placentae and breast milks were investigated. Haemolysates were prepared by freezing and thawing packed washed red cells. Aqueous placental extracts were prepared by homogenizing 1 g of tissue in 2 ml distilled water, followed by centrifugation a t 1 O O O O g for 15 min a t 4 "C. The clear supernatants were used for immunoreactivity studies. Milk samples were prepared by centrifugation a t 1000 g for 15 min. The lower (non-fat)layer was collected for study. Antibodies
Polyclonal antibodies to purified rabbit muscle phosphoglucomutase were raised in a sheep using a standard protocol (Whitehouse et al. 1989). An IgG fraction of the antiserum was prepared by affinity chromatography using a 5-0 ml Sepharose-protein G column as directed by the manufacturer (Pharmacia). Antibody treatments
Various aqueous dilutions of an IgG fraction of the antiserum were mixed with neat haemolysate, placental extract or defatted milk and incubated a t 4 "C for 4 h. The total reaction volume used was between 100 and 200 ,ul according to the experiment. This material was used for analysis without further modification. I n all experiments the untreated control samples were adjusted to the same dilution as the treatments with distilled water, Electrophoresis
Starch gel electrophoresis was conducted using a Tris-EDTA-maleate buffer system (Harris & Hopkinson, 1976). Equilibrium polyacrylamide gel isoelectric focusing was performed using
a pH 5-7 gradient (Whitehouse et al. 1989). The zones of PGM activity were detected using a specific reaction mixture applied to the gel in an agar overlay (Harris & Hopkinson, 1976).
Antigenic analysis of PGM
265
4PGM2 PGMl
-Hb
1
2
3
4
5
Fig. 1. Starch gel electrophoresis showing immunoprecipitation of PGMl in a haemolysate (phenotype PGMl 1) with various dilutions of IgG as follows: (2) 1 in 2 ; (3) 1 in 3 ; (4)1 in 4;(1) and ( 5 ) are untreated controls. The positions of the normal P G M l and PGM2 isozymes are indicated.
RESULTS
PGMl The PGM isozyme patterns of human haemolysates following incubation with various dilutions of an anti-rabbit PGMl IgG fraction were analysed by starch gel electrophoresis and enzyme activity staining. It can be seen that PGMl was completely inactivated by reaction with an IgG dilution of 1 in 2 (Fig. 1 ) . It seems most likely that inactivation was due to immunoprecipitation, since the IgG precipitated human skeletal muscle PGM in Ouchterlony double diffusion plates. One weakly staining precipitin line was produced which fused with the major precipitin line produced by interaction of the IgG with purified rabbit muscle PGM, without forming a spur in either direction. At higher IgG dilutions ( 1 in 3 and 1 in 4,Fig. l ) , a single component of PGM activity was observed that migrated more slowly than the PGMl isozymes in the untreated controls. This component was of consistently lower activity than the control PGMl and was probably due to the presence of unprecipitated PGMl enzyme-antibody complexes. To investigate the reaction of the antibodies with the PGMl isozymes by immunobinding as opposed to liquid immunoprecipitation, several 0 5 ml protein G Sepharose columns were prepared in modified 1.5 ml microcentrifuge tubes. 250 ,ul of neat anti-rabbit PGMl antiserum was loaded on to each column which was then washed with 0.05 M-Na phosphate buffer (pH 7-0) to remove unbound material. The resulting protein G-anti-rabbit PGMl IgG column was employed for immunoaffinity chromatography. 250 ,ul of haemolysate was applied to the column and the unbound fraction collected by centrifugation a t 800 g for 5 min and analysed 19 2
G. A. DRAGO AND
266
OTHERS
+ PGM2 PGMl
-Hb
+ PGMl phenotype
1
+
3
2-1
+
1
-
+ 2-1
Fig. 2. Starch gel electrophoresis showing PGM isozyme patterns from four haemolysates before ( - ) and following ( + ) immunoaffinity chromatography. Experiments with two PGMl phenotypes (1 and 2-1) are illustrated.
using starch gel electrophoresis. Figure 2 shows that the immunoaffinity columns removed all of the PGMl activity in the samples. There was no trace of the slowly migrating isozymes(s) seen in the previous experiment with the higher IgG dilutions (Fig. 1). Seventeen haemolysates representing four common PGMl phenotypes (1+, l+l-,2-1+, 2+l+) were tested for crossreaction by incubating 80 p1 of haemolysate with 20 pl of undiluted IgG fraction (1 in 5) and analysed by isoelectric focusing, gradient pH 5-7 (Fig. 3). In each case the PGMl isozyme activity was completely abolished. Thus it was concluded that there were no major differences in the antigenic properties of the products of the four common alleles ( l + ,1-, 2+, 2-) found in haemolysates. This is in keeping with immunoblotting studies where the IgG was shown to react with the PGMl isozymes of the common phenotypes immobilized on nitrocellulose filters (Whitehouse et al. 1989).
PGM2 It is evident from the liquid immunoprecipitation experiments that the human PGM2 isozymes in the haemolysates were completely nonreactive with the anti-rabbit PGMl antibodies, even a t the lowest dilution of the IgG fraction employed (Figs 1 and 3). Furthermore, the PGM2 isozymes passed through the immunoaffinity columns without marked alteration (Fig. 2). This suggests that the PGMl and PGM2 proteins are antigenically distinct.
Antigenic analysis of PGM
267
acidic
basic
+ PGMl phenotype:
-
2+1+
+ - + - + - + 2+1+ 2+1+
1+1-
-
2-1+
+ - + - + 1+1’
2-1+
2+1+
Fig. 3. Isoelectric focusing analysis of erythrocyte PGM in a pH 5-7 gradient before ( - ) and following ( + ) treatment with a 1 in 5 dilution of IgG. Three common PGMl subtypes are illustrated.
basic
*
c
Fig. 4. Isoelectric focusing in a pH 5-7 gradient showing erythrocyte PGM2 isozyme patterns from unrelated individuals following removal of PGMl activity by immunoaffinity chromatography. The possible variant phenotype is indicated (*). An untreated control sample (C), phenotype PGM 1+1-, is included for comparison.
G. A. DRAGO AND
268
OTHERS
+
PGM3 PGM2 PGMl
1
2
3
Fig. 5. Starch gel electrophoresis showing the PGM isozymes in a placental extract following treatment with various dilutions of IgG as follows: (2) 1 in 2; (3) 1 in 3 ; (1) is an untreated control. The PGM2 and PGM3 isozymes are unaffected by the treatment with IgG. Prolonged incubation, leading to overstaining of PGMl and PGM2, was necessary to visualize the PGM3 isozymes.
The capacity of the IgG to remove all of the PGMl activity from the pattern provided a novel opportunity of conducting an unambiguous assessment of the PGM2 pattern on high resolution isoelectric focusing gels. This analysis was performed using a small population sample (n = 32) to determine if hitherto undiscovered common genetic variation a t the PGM2 locus could be detected. As anticipated, treatment of the samples either with a 1 in 5 dilution of IgG or by using immunoaffinity chromatography, resulted in the complete removal of the PGMl activity from the samples. Following isoelectric focusing in gradient p H 5-7 gels and activity staining, a complex series of PGM2 isozymes was resolved (Figs 4 and 3). No proven genetically determined person-to-person variation was found in this small sample. A single sample, from a north European individual, was found to have a variant PGM2 pattern which could have been heterozygous. Unfortunately, it was not possible to obtain a repeat blood sample from this person or to conduct a family study. Nonetheless, the approach of utilizing the IgG as a reagent to remove ‘contaminating’ PGMl components is likely to be a useful adjunct in further isoelectric focusing studies of PGM2 variation.
PGM3 The immunoreactivity of the human PGM3 isozymes was investigated using placental extracts as a source of the enzyme. Following incubation of extracts with a 1 in 3 dilution of the IgG fraction and starch gel electrophoresis, it can be seen that there was no crossreaction with
Antigenic analysis of PGM
269
+
1
2
3
4
5
6
7
8
9
Fig. 6. Starch gel electrophoresis showing PGM4 isozymes in untreated milk samples from two mothers (AG and CL) and complete removal of PGM4 activity by immunoprecipitation using various IgG dilutions: (3) AG untreated; (4) AG 1 in 5 ; ( 5 ) AG 1 in 10; (6) AG 1 in 20; ( 7 ) CL untreated; (8) CL 1 in 5 ; (9) CL 1 in 10. A haemolysate, before ( 1 ) and after (2) IgG treatment, is also illustrated.
the PGM3 isozymes (Fig. 5 ) . Complete removal of normal PGMl activity from the placental extract was achieved with all antibody dilutions and, as in the previous experiments, the PGM2 activity was undiminished. Interestingly, the amount of slowly migrating enzyme-antibody complexed material was more abundant in these placental extracts than in the haemolysates. This reflects the relatively high level of PGMl activity in placenta. It can be seen that the activity of the PGM-antibody complexes increased as the concentration of the reagent antibodies decreased.
PGM4 in milk To investigate PGM4, defatted milk sample from two mothers were incubated with various dilutions of the IgG fraction and analysed by starch gel electrophoresis and specific activity staining (Fig. 6). I n both cases the PGM4 isozyme activity was completely removed, even a t an IgG dilution of 1 in 20. This was about four times less anti-rabbit PGMl IgG than was required to remove all of the PGMl activity from haemolysates. At higher dilutions of IgG traces of PGM4 isozyme activity with normal electrophoretic mobility were observed. PGM4 isozyme patterns were also investigated by isoelectric focusing. Eighteen samples were examined before and after treatment with the IgG fraction. Considerable variation in the PGM4 isozyme pattern was identified but in every case all of the milk PGM activity was removed by treatment with the IgG (Fig. 7).
G. A. DRAGO AND
270
OTHERS
acidic
basic
-
+
+
1
+ - + - +
-
+
I
+
+
I +
I
+
I
rbc
rbc
Fig. 7 . Isoelectric focusing in a pH 5-7 gradient of PGM4 in eight milk samples before ( - ) and after ( ) treatment with a 1 in 5 dilution of IgG and two haemolysate controls (rbc). PGM4 isozyme variation is evident in the untreated milk samples.
+
DISCUSSION
Phosphoglucomutase is encoded by multiple gene loci that are thought to constitute a structurally related gene'family . We have employed polyclonal antibodies raised against rabbit muscle PGMl to investigate the antigenic relationships of human PGM1, PGM2, PGMS and PGM4. Of the well-characterized isozymes encoded by PGMl, PGMB and PGMS, only PGMl was found to be immunoreactive. Furthermore, the primary and secondary isozymes of all of the variant PGMl phenotypes examined reacted equally well with the antibodies. Thus PGMl is not immunologically related to PGM2 or PGM3. This conclusion suggests that if the three loci arose by duplication of a common ancestor gene, sufficient mutational differences have occurred to render the three sets of isozymes antigenically distinct. This level of structural divergence is in keeping with the divergent biochemical properties of the PGM isozymes (Quick et al. 1974; Fisher & Harris, 1972; McAlpine et al. 1970b, c ) . Although it is feasible that the heterospecific anti-rabbit antibodies may recognize only a small number of the potential antigenic determinants on human PGMl, it has been shown that there is 97 'YO homology between the rabbit and human PGMl derived amino-acid sequences (Whitehouse et al. 1991).Therefore i t is reasonable to assume that the majority of the PGMl antigenic determinants will be conserved between the two species. An independent study using anti-human polyclonal reagents has also produced strong evidence that the human erythrocyte PGMl and PGMB isozymes are not immunologically related (Piatti et al. 1990). The origin and properties of the unique human PGM4 isozymes are less well understood. Analysis of a putative genetic polymorphism of PGM4 suggests it is independent of the PGMl locus (Cantu & Ibarra, 1982), although family studies were not possible and heritability of the isozyme patterns could not be proved. PGM4 isozymes are found only in milk, suggesting that their regulation differs considerably from PGMl ; furthermore, milk appears t o be completely devoid of activity associated with the expression of PGM1, PGMS and PGMS. The antigenic
Antigenic analysis of PGM
27 1
similarity of the PGMl and PGM4 isozymes, taken together with their shared catalytic properties, would be in keeping with a recent gene duplication where considerable sequence homology has been preserved. However, in the absence of unambiguous inherited variation, the data are also consistent with an alternative origin of PGM4 from post-transcriptional or posttranslational processing of PGMl gene products. The method we have employed to determine the immunoreactivities of the PGM isozymes using electrophoresis is rapid, simple to perform and provides a broad qualitative assessment of antigenic properties. For instance the reactivities of both primary and secondary isozymes as well as a range of phenotypes of PGM can be assessed in a single experiment. However, the determination of features such as degree of antibody binding would be better accomplished using a more quantitative technique such as ELISA or enzyme assay. It is expected that the immunological approach will continue to provide useful information on structural features differentiating the isozymes encoded by the members of the human PGM gene family. It will be particularly interesting to investigate the reactions of monoclonal antibodies to determine if individual epitopes associated with linear regions of the protein vital for catalytic activity have been conserved by all the members of the gene family. G. A. Drago was supported by a grant from the Home Office.
REFERENCES
CANTU,J. M. & IBARRA, B. (1982).Phosphoglucomutase : evidence for a new locus expressed in milk. Science 216, 639-640. CARTER,N. D., WEST, C. M., EMES, E., PARKIN, B. & MARSHALL,W. H. (1979). Phosphoglucomutase polymorphism detected by isoelectric focusing : gene frequencies, evolution and linkage. Ann. Hum. Biol. 6, 22 1-230. FISHER, R. A. & HARRIS,H. (1972). ‘Secondary’ isozymes derived from the three PGM loci. Ann. Hum. Genet. 36, 69-77. HARRIS,H. & HOPKINSON, D. A. (1976). Handbook of Enzyme Electrophoresis in Human Genetics, 2.7.5.1, 1. Amsterdam : North-Holland Publishing Co. MCALPINE,P. J., STRANC, L. C., BOUCHEIX, C. & SHOWS, T. B. (1990).The 1990 catalog of mapped genes and report of the nomenclature committee. Cytogenet. Cell Genet. 5 5 , 5-76. MCALPINE,P. J . , HOPKINSON, D. A. & HARRIS,H. (1970a). Molecular size estimates of human phosphoglucomutase isozymes by gel filtration chromatography. Ann. Hum. Genet. 34, 177-185. MCALPINE,P. J., HOPKINSON, D. A. & HARRIS, H. (1970b).Thermostability studies on the isozymes of human phosphoglucomutase. Ann. Hum. Genet. 34, 61-71, MCALPINE, P. J., HOPKINSON, D. A. & HARRIS,H. ( 1 9 7 0 ~ )The . relative activities attributable to the three phosphoglucomutase loci (PGM,, PGM, and PGM,) in human tissues. Ann. Hum. Genet. 34, 169-175. PIATTI,E., ACCORSI, A,, PIACENTINI, M. P. & FAZI,A. (1990). Immunological studies on erythrocyte phosphoglucomutase isozymes. Biochem. Int. 21, 155-163. QUICK,C. B., FISHER, R. A. & HARRIS,H. (1974). A kinetic study of the isozymes determined by the three phosphoglucomutase loci PGM,,PGM, and PGM,.Europ. J. Biochem. 42, 511-517. RAY,W. J., JR& PECK, E. J., J R . (1972).Phosphomutases. I n The Enzymes, vol. 6, 3rd edn (ed. P. D. Boyer), pp. 407-477. New York, London: Academic Press. QENSABAUGH, G. F. & CRIM,D. (1982).Biochemical markers of individuality. I n Forensic Science Handbook (ed. R. Saferstein), pp. 338415. New Jersey : Prentice-Hall Inc. N., HOPKINSON, D. A. & HARRIS,H. (1964). Phosphoglucomutase polymorphism in man. Nature 204, SPENCER, 742-745. WHITEHOUSE,D. B., DRAGO,G. A,, HOPKINSON,D. A,, WESTWOOD,S. A. & WERRETT,D. J. (1989). Immunological detection of human phosphoglucomutase (PGM1) subtypes. Forensic Sci. Int. 41, 25-34. WHITEHOUSE, D. B., PUTT, W., LOVEGROVE, J . U., MORRISON, K., HOLLYOAKE, M., Fox, M., HOPKINSON, D. A. & EDWARDS, Y. H. (1991).Phosphoglucomutase 1 (PGMl); complete human and rabbit mRNA sequences and direct mapping of this highly polymorphic marker on human chromosome 1. Proc. Natl. Acad. Sci.USA (in press).
