Human Hypoxanthine (Guanine) Phosphoribosyltransferase: An

Proc. NatL Acad. Sci. USA Vol. 80, pp. 870-873, Febriary 1983 Medical Sciences

Human hypoxanthine (guanine) phosphoribosyltransferase: An amino acid substitution in a mutant form of the enzyme isolated from a patient with gout (reverse-phase HPLC/peptide mapping/mutant enzyme) JAMES M. WILSON*t, GEORGE E. TARRt, AND WILLIAM N. KELLEY*t Departments of *Internal Medicine and tBiological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109 Communicated by James B. Wyngaarden, November 3, 1982 ABSTRACT We have investigated the molecular basis for a tration ofenzyme protein. in both erythrocytes (3) and lympho- deficiency ofthe enzyme hypoxanthine (guanine) phosphoribosyl- blasts (4); (ii) a normal Vm., a normal Km for 5-phosphoribosyltransferase (HPRT; IMP:pyrophosphate-phosphoribosyltransfer- 1-pyrophosphate, and a 5-fold increased Km for hypoxanthine ase, EC 2.4.2.8) in a patient with a severe form of gout. We re-. (unpublished data); (iii) a normal isoelectric point (3, 4) and ported in previous studies the isolation of a unique structural migration during nondenaturing polyacrylamide gel electro- variant of HPRT from this patient's erythrocytes and cultured phoresis (4); and (iv) -an apparently smaller subunit molecular lymphoblasts. This enzyme variant, which is called HPRTOnd0., weight as evidenced by an increased mobility during Na- is characterized by a decreased concentration of HPRT protein DodSO4/polyacrylamide gel electrophoresis (3, 4). in erythrocytes and lymphoblasts, a normal Vm.., a 5-fold in- Our study ofthe tryptic peptides and amino acid composition creased Km for hypoxanthine, a normal isoelectric point, and an of apparently smaller subunit molecular weight. Comparative pep- HPRTLondon revealed a single amino acid substitution (Ser tide mapping-experiments revealed a single abnormal tryptic pep- Leu) at position 109. tide in HPRTondon. Edman degradation of the aberrant peptide from HPRT~ondon identified a serine-to-leucine amino acid sub- EXPERIMENTAL PROCEDURES stitution at position 109. This substitution can be explained by a HPRT Purification, Trypsin Digestion, and Peptide Map- single nucleotide change in the codon for. serine-109 (UCA ping. HPRT was purified from erythrocytes obtained from nor- UUA). Thus a mutation at the HPRT locus has now been defined mal subjects and from a patient with the structurally altered at the molecular level. enzyme HPRTLondon. The enzyme purifications were performed according to published procedures (3, 7). In preparation A complete deficiency of hypoxanthine (guanine) phosphori- fordigestion with trypsin, the purified enzymes were denatured bosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyl- in 6 M guanidine-HCI and S-pyridylethylated (8). The pyridyl- transferase, EC 2.4.2.8) is associated with the Lesch-Nyhan ethylated enzymes (20 nmol of normal HPRT and 10 nmol of syndrome (1), whereas a partial deficiency ofthe same enzyme HPRTLondon) were digested with trypsin (1% by weight) as de- leads to purine overproduction and gout (2). The precise mu- scribed (6). The trypsin digests were fractionated and the com- tational events that cause these X-linked enzyme deficiency plex peaks were resolved by reverse-phase HPLC as described states. have remained undefined. One possible mechanism is (6). a mutation within the coding sequences ofthe HPRT structural Amino Acid Composition and Sequence Analysis. The intact gene that leads to the synthesis of a structurally altered HPRT pyridylethylated enzymes were hydrolyzed in 6 M HCl for 24 protein. Direct evidence in support of-this hypothesis was pro- hr and were analyzed for amino acid content as described (5). vided by the isolation offive unique structural variants ofHPRT Peptide fragments were subjected to manual Edman degrada- from unrelated HPRT-deficient patients (3, 4). tion using the methods described by Tarr (9, 10). The resulting Recent advances made in our understanding ofthe structure phenylthiohydantoin amino acids were analyzed by HPLC (11). of the normal human HPRT enzyme have made it possible to begin a detailed investigation of the nature and consequences of mutations in its structural gene. We have recently defined RESULTS the complete amino acid sequence ofthe normal human enzyme Purification and Amino Acid Composition of Normal HPRT (5). The enzyme is 217 residues long and undergoes two post- and HPRTIondn. Normal HPRTwas purified from pooled nor- translational modifications in erythrocytes: acetylation of the mal hemolysate (330 individual units ofnormal blood) and from NH2-terminal alanine (5) and partial deamidation ofasparagine hemolysate obtained from a single normal male subject. These 106 (6). In addition, a sensitive method for mapping the tryptic apparently normal enzymes were indistinguishable in terms of peptides ofthe normal human enzyme has been developed (6). their subunit molecular weights (3), isoelectric points (3), and In the present study, we have attempted to define the tryptic peptide patterns (data not shown). All subsequent stud- molecular abnormality in the previously described variant ies of normal HPRT were performed on the enzyme purified HPRTLondon. This unique structural variant was purified from from pooled hemolysate.. HPRTLondon was purified with a 23% erythrocytes (3) and lymphoblasts (4) ofa male patient who pre- recovery, of enzyme activity from erythrocytes (500 ml) of pa- sented with hyperuricemia and an early onset ofgout. Previous tient G. S. (3). NaDodSO4/polyacrylamide gel electrophoresis studies of the function and subunit structure of HPRTLndon indicated that the normal and mutant enzyme preparations demonstrated the following properties: (i) a decreased concen- were greater than 95% pure. The purified enzymes were pyridylethylated and a portion The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviation: HPRT, hypoxanthine (guanine) phosphoribosyltransfer- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. ase. 870 Downloaded by guest on September 28, 2021 Medical Sciences: Wilson et aL Proc. NatL Acad. Sci. USA 80 (1983) 871 of each (6%) was analyzed for amino acid composition. All res- Table 1. Amino acid composition of normal HPRT idues except alanine, glycine, and tryptophan were quantitated and HPRTI,.d.. accurately. This analysis documented atotal recovery of20 nmol Composition,* ofnormal HPRT and 10 nmol ofHPRTLOndOn. The compositions residues per subunit of the normal and variant enzymes, shown in Table 1, are in good agreement. Except for leucine and serine, individual Residue Normal HPRT HPRTL.d0n amino acids differed between the normal and mutant enzyme Asx 29.3 29.3 by less than one residue per subunit; HPRTLnd0n exhibited an Thr 10.9 11.2 increased value for leucine and a decreased value for serine. Ser 13.8 11.7 Peptide Mapping of Normal HPRT and HPRTLoIdon. In an Glx 16.0 15.6 earlier study, we described a HPLC method for mapping the Pro 9.5 10.1 tryptic peptides of normal human erythrocyte HPRT (6). We Gly NDt NDt have used this method to identify the structural alteration in Ala 10.9 NDt HPRTLndon. The pyridylethylated forms ofnormal HPRT and Val 17.2 17.7 HPRTLnd0. were digested with trypsin for 6 and 18 hr, and the Met 5.7 5.8 digests were resolved by reverse-phase HPLC. Representative fle 12.0 11.8 chromatograms of these separations are shown in Fig. 1. Leu 20.8 21.8 Although most peaks remained unchanged after 6 hr oftryp- Tyr 9.8 9.1 sin digestion, several later-eluting peaks from both enzymes Phe 7.9 7.1 exhibited reproducible, time-dependent changes in their rel- His 4.3 4.0 ative peak height. This was shown in a previous study to be due Lys 14.5 14.3 to the slow hydrolysis ofthe Lys-Phe bond at position 185-186 Cyst 2.8 3.2 and the partial cleavage of several non-Lys/Arg bonds (6). Arg 11.8 11.4 The peptide map of normal HPRT was very similar to that Trp ND ND 6 and 18 hr of digestion. Ofparticular im- of HPRTLondon after * Values represent duplicate analyses (50% of the sample per analysis) portance was the identity of the COOH-terminal peptide (la- of a single hydrolysis of normal HPRT (1.9 nmol) and of HPRTLotd0n beled 27 in Fig. 1) in the normal and mutant enzymes. This (0.6 nmol). ND, not determined. finding effectively rules out any mutation that modifies the t Not determined because an inconsistent base line precluded an ac- structure of the COOH-terminus, such as a frameshift or pre- curate quantitation. mature chain termination mutation. The one difference in * Quantified as pyridylethylcysteine.

0.07 | NORMAL HPRT- 18 HOURS 60

0.06- 50 - 0.06 257 r E 0.05 C 0.0,5 ) _ w -j 0.04 0.04- 4.t 30: w 4 0 0.03 ' 0.03- 140 a4 ~~ iA~~~~~hs ~~~20 Il w iY - 7 0 0.02 ~002 i I / U F 2I 4 nni I

I0.06

E 0.05 c l I

.-1 ti 0.04 E <, 0.03 2 0

<0 QO 4 (0 at 941 0.0I1

30 40 50 30 40 50 TIME (MINUTES) TIME (MINUTES) and FIG. 1. Tryptic peptide maps of normal HPRT and HPRTLO,&On. Trypsin digests were lyophilized, resuspended in 5% (vol/vol) formic acid, fractionated by reverse-phase HPLC. These chromatograms represent analytical separations of a portion (300-400 pmol) of each digest: normal HPRT and HPRTIond0. digested with trypsin for 6 hr and 18 hr. Preparative separations were performed under identical conditions with nearly identical peptide profiles. Downloaded by guest on September 28, 2021 872 Medical Sciences: Wilson et aL Proc. Nad Acad. Sci. USA 80 (1983)

Table 2. Manual Edman degradation of peptide 14 from normal at position 109 has shifted the retention times ofpeptides 14 and HPRT and peptide 14' from HPRTLOndon* 14d from 19.5 min and 19.8 min in normal HPRT to 27.1 min Peptide 14, Peptide 14', and 27.7 min in HPRTLondon. These results are summarized in normal HPRT HPRTLondon Fig. 2. Cycle Positiont Residue Yield, nmol Residue Yield, nmol DISCUSSION 1 103 Ser 1.85 Ser 1.13 We have defined the molecular abnormality in a structural vari- 2 104 Tyr 1.12 Tyr 0.61 ant ofhuman HPRT (HPRTLondon) isolated from a patient with 3 105 Cys 1.06 Cys 0.38 a severe form of gout. These studies were complicated by the 4 106 Asn 1.08 Asn 0.61 limited quantity ofpurified HPRTLondon that was available; ap- 5 107 Asp 0.80 Asp 0.44 proximately 250 Ag of HPRTLondon was isolated from 500 ml of 6 108 Gln 0.72 Gln 0.33 erythrocytes. A detailed analysis of the primary structure of 7 109 Ser 0.70 Leu 0.21 HPRTLondon, therefore, required sensitive methods of peptide 8 110 Thr 0.53 Thr 0.25 mapping and protein sequence analysis. The tryptic peptides 9 111 Gly 0.46 Gly 0.23 were mapped and purified by reverse-phase HPLC (6) and the 10 112 Asp 0.30 Asp 0.14 sequences ofpeptide fragments were determined by using the 11 113 Ile 0.13 Be 0.04 micromethods ofmanual Edman degradation described by Tarr 12 114 Lys 0.08 Lys 0.03 (9, 10). * A portion of each purified peptide (35% of 14 and 50% of 14') was A detailed analysis of the tryptic peptides of HPRTLondon re- subjected to Edman degradation. Only one amino acid was released vealed an amino acid substitution (Ser -+ Leu) at position 109. at each cycle of these degradations. We have reported 10 cycles of Evidence in support ofthis substitution was provided by amino Edman degradation on peptide 14' with identical results. acid analysis of the intact enzyme. Recent studies on the nu- t Indicates the position of the amino acid with respect to the NH2 ter- cleotide sequence ofhuman HPRT cDNA have established the minus of the intact enzyme (5). codon for Ser-109 as UCA (12). The serine-to-leucine substitution in HPRTLondon can be explained by a single nucleotide HPRTLondon that was noted at both time points was the absence change in this codon (UCA -- UUA). ofpeptides 14 and 14d and the appearance ofa new peak at 27.1 In a previous study, we demonstrated that each tryptic pep- min (labeled 14' in Fig. 1). tide predicted from the amino acid sequence ofhuman HPRT Peptides that eluted as mixtures (peaks at 4.3, 8.2, 30.3, and is recovered in high yield from the reverse-phase HPLC sep- 58.0 min in Fig. 1) were rechromatographed under different aration (6). This approach to peptide mapping, therefore, as- conditions to screen for alterations that may have been obscured sured a direct comparison of peptides that span the entire en- in the original peptide map. The component peptides of these zyme molecule. We conclude that a second site of mutation in complex peaks were resolved by reverse-phase HPLC with a HPRTLondon is highly unlikely because, with the exception of different column (6). These experiments detected no additional peptides 14 and 14d, the tryptic peptide pattern ofHPRTLondon alterations in the chromatographic properties of the peptides was indistinguishable from the pattern of the normal enzyme. in HPRTLondon as compared to the corresponding peptides of Further evidence against a second structural alteration in the normal enzyme (data not shown). HPRTLondon was provided by rechromatographing the peaks Amino Acid Sequence ofAberrant Peptides in HPRTLnd0n. from the original peptide map on a different reverse-phase col- The entire sequences of peptide 14 from normal HPRT and umn. No additional chromatographic abnormalities were de- peptide 14' from HPRTLondon were established by manual Ed- tected in the peptides from HPRTLondon in these experiments. man degradation. As shown in Table 2, these peptides differ by HPRTLond0n is indistinguishable from normal HPRT in terms a single amino acid at position 109; a serine in normal HPRT ofits isoelectric point and migration duringnondenaturing poly- and a leucine in HPRTLond0. Peptide 14d differs from peptide acrylamide gel electrophoresis (3, 4). These findings are con- 14 by the presence ofan aspartic acid in place ofan asparagine sistent with the proposed neutral-to-neutral amino acid substi- at position 106, reflecting partial deamidation of the normal tution. A finding that was apparently inconsistent with an amino HPRT protein in vivo (6). The corresponding peptide in acid substitution was the faster migration that HPRTLondon ex- HPRTLondn, peptide 14', eluted to the right ofpeptide 14' as hibited during NaDodSO4/polyacrylamide gel electrophoresis; a mixture with peptide 25. The NH2-terminal sequence ofpep- the apparent subunit molecular weight of HPRTLondon was tide 14' was established by subjecting the mixture to seven decreased in comparison to the normal value by the equiv- cycles ofEdman degradation and subtracting the sequence con- alent of 8-10 amino acid residues (3, 4). Our studies of the tributed bypeptide 25 (data not shown). This analysis confirmed tryptic peptides and amino acid composition of HPRTLondon the serine-to-leucine substitution at position 109 as well as the argue against a real difference in the length of this variant en- deamidation of asparagine-106. zyme. A secondary change in the subunit molecular weight of These studies indicate that a serine-to-leucine substitution HPRTLondon due to differential processing (i.e., glycosylation NORMAL HPRT HPRTLondon

105 A 110 115 110 115 e Lys Ser Tyr Cys As' Asp GIn Ser Thr Gly Asp Ile Lys Val - -*--Lys Ser Tyr CysAsn105,h-n Asp Gln Leu Thr Gly Asp lie Lys Val-. A AAsp A ApA Trypsin Trypsin Tpl4 Ser Tyr Cys Asn Asp GIn Ser Thr Gly Asp Ile Lys Tpl4 Ser Tyr Cys Asn Asp GIn Leu Thr Gly Asp lie Lys Tpl4d Ser Tyr Cys Asp Asp Gin Ser Thr Gly Asp Ile Lys Tp14j Ser Tyr Cys Asp Asp GIn Leu Thr Gly Asp lie Lys

FIG. 2. Peptides 14 and 14d from normal HPRT and the corresponding peptides from HPRTLondon. Tp, tryptic peptide. The arrowheads indicate bonds that are cleaved by trypsin. Downloaded by guest on September 28, 2021 Medical Sciences: Wilson et aL Proc. Natd Acad. Sci. USA 80 (1983) 873

is also unlikely (3). A more likely explana- The fine technical assistance of Laurie Landa and Ryuji Kobayashi or phosphorylation) S. and his family for their tion for the anomalous migration of HPRTLund0n during Na- was greatly appreciated. We thank patient G. is that the serine- cooperation, and Dr. I. Fox for his help in obtaining the blood samples. DodSO4/polyacrylamide gel electrophoresis We also thank Drs. D. Jolly and T. Friedmann for communicating un- to-leucine substitution has increased the binding ofNaDodSO4 published results. This work was supported by Grant R01-AM19045 to the enzyme, resulting in its faster electrophoretic mobility. from the National Institutes of Health. J. M.W. is a fellow in the Medi- There are, indeed, previous examples ofsingle amino acid sub- cal Scientist Training Program. stitutions that have affected a protein's migration during NaDodSO4/polyacrylamide gel electrophoresis (13, 14). de 1. Seegmiller, J. E., Rosenbloom, F. M. & Kelley, W. N. (1967) Jong et al suggest that hydrophilic-to-hydrophobic amino acid Science 155, 1682-1684. F. & such as the serine-to-leucine substitution in 2. Kelley, W. N., Rosenbloom, F. M., Henderson, J. Seeg- substitutions, Proc. Acad. Sci. USA 57, 1735-1739. are more likely to increase NaDodSO4 binding, miller, J. E. (1967) Nati HPRTLondon, 3. Wilson, J. M., Baugher, B. W., Landa, L. & Kelley, W. N. giving the resultant increased electrophoretic mobility (13). (1981) J. Biol Chem. 256, 10306-10312. Mutant forms of HPRT have also been isolated from muta- 4. Wilson, J. M., Baugher, B. W., Mattes, P. M., Daddona, P. E. genized mammalian somatic cells selected for HPRT deficiency & Kelley, W. N. (1982) J. Clin. Invest. 69, 706-715. (reviewed in ref. 15). Many ofthese abnormal enzymes exhib- 5. Wilson, J. M., Tarr, G. E., Mahoney, W. C. & Kelley, W. N. gel (1982)J. Biol. Chem. 257, 10978-10985. ited altered mobilities during NaDodSO4/polyacrylamide W. N. This type of electrophoretic abnor- 6. Wilson, J. M., Landa, L. E., Kobayashi, R. & Kelley, electrophoresis (16-20). (1982) J. Biol Chem. 257, 14830-14834. mality was used as evidence to suggest mutations that affect the 7. Holden, J. A. & Kelley, W. N. (1978) J. Biol Chem. 253, 4459- length of the HPRT polypeptide (i.e., nonsense mutations, 4463. deletions, or mutations that modify proteolytic processing). Our 8. Hermodson, M. A., Ericsson, L. H., Neurath, H. & Walsh, K. studies on HPRTLond0n provide structural evidence for a single A. (1973) Biochemistry 12, 3146-3153. causing an artefac- 9. Tarr, G. E. (1977) Methods Enzymol. 47, 335-357. amino acid substitution in an HPRT variant ed. in its apparent subunit molecularweight. We con- 10. Tarr, G. E. (1982) in Methods in Protein Sequence Analysis, tual decrease Elzinga, M. (Humana, Clifton, NJ), pp. 223-232. clude that an abnormal migration during NaDodSO4/poly- 11. Black, S. D. & Coon, M. J. (1982) AnaL Biochem. 121, 281-285. acrylamide gel electrophoresis does not necessarily indicate an 12. Jolly, D. J., Okayama, H., Berg, P., Esty, A. C., Filpula, D., alteration in the length of the HPRT protein. In addition, Bohlen, P., Johnson, G. G., Shively, J. E., Hunkapillar, T. & NaDodSO4/polyacrylamide gel electrophoresis may prove to Friedmann, T. (1983) Proc. Natl Acad. Sci. USA 80, 477-481. be an effective electrophoretic technique for detecting neutral 13. de Jong, W. W., Zweers, A. & Cohen, L. H. (1978) Biochem. amino acid substitutions in other structural variants of human Biophys. Res. Commun. 82, 532-539. 14. Noel, D., Nikaido, K. & Ames, G. F. (1979) Biochemistry 18, HPRT. 4159-4165. In summary, we have identified the molecular abnormality 15. Caskey, C. T. & Kruh, G. D. (1979) Cell 16, 1-9. in HPRTLondon, a serine-to-leucine substitution at residue 109. 16. Milman, G., Krauss, S. W. & Olsen, A. S. (1977) Proc. Natl This structural alteration results in an increased Km for hypo- Acad. Sci. USA 74, 926-930. xanthine and a decreased intracellular concentration of HPRT 17. Capecchi, M. R., Vonder Haar, R. A., Capecchi, N. E. & Sveda, protein, both effects contributing to the deficiency of enzyme M. M. (1977) Cell 12, 371-381. relationship 18. Fenwick, R. G., Jr., Sawyer, T. H., Kruh, G. D., Astrin, K. H. activity in vivo. A complete understanding of the & Caskey, C. T. (1977) Cell 12, 383-391. between the amino acid substitution and the kinetic abnor- 19. Epstein, J., Ghangas, G. S., Leyva, A., Milman, G. & Little- mality and decreased intracellular enzyme concentration will field, J. W. (1979) Somatic Cell Genet. 5, 809-820. require additional studies ofthe enzyme's intracellular turnover 20. Kruh, G. D., Fenwick, R. G., Jr., & Caskey, C. T. (1981)J. Biol and three-dimensional structure. Chem. 256, 2878-2886. Downloaded by guest on September 28, 2021