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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-phosphoribosyl- transferase (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 (unpublished data); (iii) a normal isoelectric point (3, 4) and ported in previous studies the isolation of a unique structural migration during nondenaturing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 per- formed 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.
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