ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 283, No. 1, November 15, pp. 120-129,199O

Isolation and Characterization of Human Glucose-6Phosphate lsomerase Isoforms Containing Two Different Size Subunits An-Qiang

Sun, K. urnit

Yiiksel, Tony M. Jacobson, and Robert W. Gracyl

Department

of Biochemistry,

University

of North

TexasjTexas

College of Osteopathic Medicine,

Fort Worth, Texas 76107

Received May 24,1990, and in revised form July 20,199O

Previously undetected isoforms of human glucose-6phosphate isomerase (GPI) have been isolated utilizing substrate-induced elution of the enzyme from spherical cross-linked phosphocellulose as an affinity ligand and subjected to a series of physical and chemical studies. The two major isoforms (1, 48%, pI 9.13; 2, 36%, pI 9.00) are homodimers of subunits of 63.2 kDa (Type-A) and are charge isomers, probably representing deamidation of specific Asn-Gly sequences as in other species. Isoform 3 (13%, pI 8.84) is a heterodimer composed of the Type-A subunit and a previously unreported larger subunit of 69.8 kDa (Type-B). Isoform 4 (3%, pI 8.62) is a BB-homodimer. Structural differences in the two types of subunits are also apparent from CNBr fragmentation patterns. Carbohydrate analyses show that, even though potential N- and O-linked glycosylation sites exist, the isoforms are not due to glycosylation. Recently recognized sequence similarities between GPI and the neurotropic lymphokine, neuroleukin (NLK) suggest that GPI and NLK are either derived from the same gene or represent modifications of the same protein. The possibility of NLK-GPI dimers exists, but the new isoforms identified in this study do not appear to represent hybrids of GPI subunits with mature NLK. o 1990 Academic Press, I~C.

Glucose-6-phosphate the least characterized

isomerase (GPI,2 EC 5.3.1.9) is of the glycolytic enzymes. This is

i To whom correspondence should be addressed at Department of Biochemistry, University of North Texas/Texas College of Osteopathic Medicine, 3500 Camp Bowie Blvd., Fort Worth, TX 76107. Fax: (817) 735-2283. ’ Abbreviations used: BME, 2-mercaptoethanol; BSA, bovine serum albumin; CAPS, 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid, DNase I, deoxyribonucleate 5’-oligonucleotide hydrolase; EC 3.1.21.1; GPI, glucose 6-phosphate isomerase; EC 5.3.1.9; IEF, isoelectric focusing; NLK, neuroleukin; PAD, pulsed amperometric detector;

120

in part due to its relatively large subunit size, a blocked amino terminus, and the general tendency of researchers to study regulated enzymes as opposed to housekeeping enzymes. Recently, the unexpected structural homology of GPI with neuroleukin (NLK), a protein exhibiting both neurotropic and lymphokine activity (l-4), has drawn attention to GPI. It has been suggested that NLK and GPI may be derived from the same gene or represent modifications of the same protein product (3, 5, 6) and that specific cleavage of GPI might be responsible for the conversion of GPI to the cancer-associated variants of GPI exhibiting the same molecular weight as NLK (7). Our laboratory initially isolated human erythrocyte GPI to homogeneity by utilizing substrate to specifically elute the enzyme from phosphocellulose (8). This method has been quite successful and has been utilized for the isolation of the enzyme from other sources (9, 10). In the course of characterizing GPI from different species and tissues, a variety of isoforms have been observed. The molecular bases for these isoforms include deamidation (ll), oxidation (10, 12), and proteolytic modification (7). A question of particular interest to our laboratory has been the basis for the accumulation of modified forms of the enzyme in aging cells and tissues. For example, we have observed that specific deamidations at Asn-Gly sequences of the enzyme appear to occur in vivo, and that the deamidated isoforms accumulate in aging cells and tissues including the eye lens (13). The initial goal of this study was to develop methods for the quantitative isolation of the protein which avoid the selective loss of such modified isoforms and minimize

PAGE, polyacrylamide gel electrophoresis; PAS, periodic acid-Schifi, PNGase F, endoglycosidase F/N-glycosidase F mixture, Boehringer Mannheim; PVDF, polyvinylidenedifluoride; RNase B, ribonuclease B; EC 3.1.27.5; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline; TEA, triethanolamine.

0003-9861/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

HUMAN

GLUCOSE-6-PHOSPHATE

the potential for modifications of the protein during isolation. The studies reported here utilize a new isolation procedure for human GPI which is much more rapid and mild, and results in improved overall recovery of the enzyme. The new procedure has led to the discovery of previously unrecognized isoforms of GPI. Thus, an additional goal has been the characterization of the molecular basis of these isoforms. The new isoforms are less stable than “native” GPI, are dimers composed of two subunits of different sizes and, based on structural analyses, represent a form of GPI which has not been previously characterized. EXPERIMENTAL

PROCEDURES

Materials. Cellulose phosphate (fibrous powder lot No. 89C-0148) was obtained from Sigma while cross-linked spherical particle cellulose phosphate (Control No. 03112) was from Serva Fine Biochemicals Inc. Rabbit anti-human GPI antisera was obtained as described by Purdy et al. (14). Peroxidase-conjugated IgG fraction of goat anti-rabbit IgG was obtained from Cooper Biomedical Inc., while reagents for automated Edman degradation were purchased from Applied Biosysterns Inc. Endoglycosidase F/N-glycosidase F mixture (PNGase F) was from Boehringer-Mannheim (Cat. No. 878740). All other reagents were of the highest purity grade available and were obtained from various manufacturers. Enzyme andprotein assays. The catalytic activity of GPI was measured in the direction of fructose 6-phosphate + glucose 6-phosphate using a coupled enzyme assay (9). The assay mixture (1 ml) contained 50 mM triethanolamine buffer, pH 8.3,4 mM fructose 6-phosphate, 0.5 mM nicotinamide adenine dinucleotide phosphate (NADP), and 1 unit of glucose-6-phosphate dehydrogenase. The reaction was followed by measuring the reduction of NADP at 340 nm. Protein content was measured according to Bradford (15). Column effluents were monitored for their absorbance at 280 nm using an extinction coefficient of E:,“, = 13.9 (16). Isolation of glucose-6-phosphate isomerase. Three to five human placentae were homogenized in extraction buffer (50 mM TEA-HCl, 5 mM EDTA, 10 mM KCl, 0.1% BME, pH 8.2) at a ratio of 150 g wet tissue per 225 ml of buffer. The homogenate was centrifuged at 9800g for 1 h at 4”C, and the supernatant solution passed through glass wool 0.1% BME, 1 mM and dialyzed against buffer A (10 mM TEA-HCl, EDTA, pH 7.2). Damp cross-linked spherical phosphocellulose, which had been cycled at least three times through 0.5 N HC1/0.5 N NaOH/ distilled water washes and preequilibrated in buffer A, was mixed with the dialyzed homogenate (1000 ml: 400 g). The mixture was washed with buffer A followed by buffer B (25 mM TEA-HCl, 0.1% BME, pH 8.2) and then poured into a chromatography column (4 X 100 cm). The column was washed with buffer B. When the effluent was protein-free as judged from the absorbance at 280 nm, the enzyme was eluted with 10 mM fructose 6-phosphate in buffer B. Electrophoretic methods. Preparative isoelectric focusing (IEF) was carried out on a LKB Multiphor system using LKB Ultrodex granules (4 g/100 ml distilled water) and Ampholines [pH 8-9.5 (3 ml), plus pH 9-11 (0.5 ml)]. The gelled matrix was prefocused at 15 W for 25 min at 4°C and the samples were resolved at 30 W for 20 h at 4°C. Preparative PAGE (BRL 1100 PG Preparative Gel Electrophoresis system; BRL, Inc.) was used to separate the two types of GPI subunits. Stacking gel (3% acrylamide, 1 cm high) and resolving gel (10% acrylamide, 4 cm high) were cast into electrophoresis tubes of 1 cm inner diameter. The running buffer contained 50 mM Tris, 0.38 M glycine, and 0.1% SDS at pH 8.2. Electrophoresis was at constant current (8 mA) at room temperature. The flow rate was 10 ml/h, and fractions of 0.73 ml were collected.

ISOMERASE

ISOFORMS

121

Nondenaturing alkaline gel electrophoresis was conducted as described by Maize1 (17). Slab gels consisted of 3.5 and 7.5% acrylamide stacking and resolving (120 mm high) gels, respectively. Electrophoresis was conducted at 4°C at 20 mA until the tracking dye entered the resolving gel, and at 30 mA thereafter. Analytical SDS-PAGE was performed as described by Laemmli (18). Polyacrylamide slabs (10% acrylamide resolving gel, 120 mm high; 3% acrylamide stacking gel; 0.1% SDS) were run at 20 mA per slab at room temperature. The proteins were visualized by Coomassie brilliant blue or silver staining (19, 20). Subunit molecular weights were determined against protein standards. Analytical isoelectric focusing (pH range of 3.5-9.5) was conducted on LKB Ampholine PAGplates at constant power (1 W/cm) with 1 M H3P04 as the anode solution and 1 M NaOH at the cathode. Apparent isoelectric points were determined relative to protein standards of known pZ values. For the pH range 8-10.5, acrylamide gel plates (0.75 X 140 X 110 mm) containing ampholytes (pH 8-11 or pH 8-10.5) were cast according to manufacturers instructions. After prefocusing for 15 min (5 W), samples were applied, the power was increased to 14 W and focused with cathode and anode solutions of 1 M NaOH and 0.25 M Hepes, respectively. Samples loaded at both ends or the center of the electric field showed no differences in the observed plvalues. Staining and analyses of gels. GPI was located after IEF or PAGE by the activity staining procedure of Snapka et al. (21). Western analyses from SDS-PAGE were done as described by Towbin et al. (22) with the following exceptions. The unoccupied sites were blocked with 3% gelatin in Tris-buffered saline (TBS; 20 mM Tris, 0.5 M NaCl, pH 7.5) for 30 min, then washed 3 X 5 min with TBS containing 0.05% Tween20. Primary antibody (rabbit anti-human GPI antisera) and secondary antibody (peroxidase conjugated goat anti-rabbit IgG) were applied at room temperature for 60 and 40 min, respectively. The blots were visualized using a color developer consisting of 20 ml of TBS, 4 ml of 4-chloro-1-naphthol stock solution (3 mg in 1 ml methanol) and 8 ~1 of 30% hydrogen peroxide. Carbohydrate analyses. Periodic acid Schiff (PAS) analyses were conducted after the proteins were resolved by SDS-PAGE (23). To visualize total protein, the Schiffs reagent was removed by washing the gel with 10% methanol/7% acetic acid, and the gel stained with Coomassie brilliant blue R (0.1% in 40% methanol/lo% acetic acid) for 30 min and destained with 10% methanol and 5% acetic acid. As an additional method to evaluate the presence of carbohydrates, the samples were treated with PNGase F as described by Tarentino et al. (24). GPI and glycoprotein standards (5 mg/ml) were prepared in 1% SDS and boiled 3 min for denaturation. PNGase F hydrolyses were conducted in 0.25 M sodium phosphate, pH 8.4, with 5 units of the enzyme to react 50 pg/lOO ~1 of each test protein. The samples were incubated at 37’C for 18 h. Carbohydrate analysis by HPLC was conducted as described by Dimitrijevich et al. (25, 26). Aqueous samples (50 ~1, 0.5 mg/ml) were analyzed on a Dionex HPLC 4000 system equipped with a CarbopackIPA- column (3.9 mm X 30 cm), an AG6 guard column, and a PAD II pulsed amperometric detector (PAD). The column was developed using a gradient of 0 to 100% eluent B in 60 min (eluent A: 1 mM NaOH, 10 mM sodium acetate; eluent B: 200 mM NaOH, 1 mM sodium acetate). The PAD was operated in the triphasic mode with 3 PA full scale. The following potentials were applied: El = 0.05 V (tl = 360 ms), E2 = 0.8 V (t2 = 120 ms), and E3 = 0.06 V (t3 = 420 ms). NaOH (0.7 M) was introduced post column at 0.5 ml/min to stabilize the detector and improve sensitivity. Amino acid analyses. After electrophoresis, proteins were electroblotted onto polyvinyledenedifluoridene (PVDF; Immobilon, Millipore) membranes and visualized by staining with Coomassie brilliant blue R-250 (27). Membranes were washed in distilled HxO (4 X 5 min) air dried and the protein bands excised. Samples were hydrolyzed (6 N HCl in uacuo, 110°C) for 24,48, and 72 h. Amino acids were quantitated by post column fluorescence detection with o-phthaldialdehyde

SUN

151 ? 10.

I

5’

A,

0

10

20

30

40

Fraction number FIG. 1. Elution profiles of GPI from different types of phosphocellulose. The dialyzed homogenate from a single placenta was divided into two portions and identical amounts of GPI activity were loaded onto two columns of the same size containing the two different kinds of phosphocellulose. GPI was eluted as described under Experimental Procedures. Fractions (150 drops/tube) were collected after the elution buffer was started. Fractions were assayed for protein concentration and GPI activity. Top panel, elution profile from cross-linked spherical particle phosphocellulose; bottom panel, from fibrous matrix. Triangles denote GPI activity, cilcles absorbance at 280 nm. The insert shows SDS-PAGE of purified human GPI after chromatography on cross-linked spherical particulate phosphocellulose. (28). Although PVDF membranes are resistant to acid hydrolysis, blanks were analyzed to assure accurate compensation for background. Values for serine and threonine were extrapolated to zero time of hydrolysis while values for the hydrophobic residues were obtained from the 72-h hydrolyses. Cyanogen bromide cleauoge and peptide isolation. Samples (0.1 mg in 50% formic acid) were cleaved at the methionine residues with CNBr (29). After lyophilization the peptides were dissolved in 25 mM TEA, pH 8.9, and analyzed on 20% SDS-PAGE. RESULTS

Isolation of human GPI. The differences in the properties of the two types of column matrices are apparent from Fig. 1. In the case of the spherical matrix, the sub-

ET AL.

strate induced elution of the enzyme in a narrow region coincident with the second (and major) protein peak, and it is superior in terms of overall purification, speed, and recovery. In contrast, when utilizing the fibrous material, the elution profile was much broader and additional proteins tended to leach from the column and interfere with ideal resolution. Table I shows that the new method resulted in about 5500-fold purification with recovery in excess of 50% and specific activity greater than 800 units/mg. We ascribe the improved results to the shortened time and mild conditions of the entire chromatographic procedure. Resolution of the isoforms. Four electrophoretic forms of GPI were observed upon isoelectric focusing and nondenaturing PAGE in a variety of electrophoretic systems at variable pore sizes and pH ranges. Each isoform was completely resolved and designated as isoforms 1, 2, 3, and 4, respectively, from the most positively (basic) to the most negatively (acidic) charged species. Isoelectric focusing was found to be the most satisfactory method for preparative isolation of the GPI isoforms. Figure 2 shows the mixture and the resolved isoforms with apparent pI values of 9.13,9.00,8.84, and 8.62. The relative amounts of each of the isozymes determined by total protein content were 48, 36, 13, and 3%, respectively. When each isoform was isolated (uide infra) and resubjected to electrophoretic or isoelectric focusing analyses, single forms were obtained (Fig. 2, lanes 3-6). The electrophoretic profiles were unchanged by the addition of reducing agents. Isolation of the subunits. When each of the isolated isoforms was subjected to SDS-PAGE as shown in Fig. 3A, two different size subunits were observed. The two more basic isozymes (1 and 2) exhibited a single band on SDS-PAGE (designated the A-type subunit) corresponding to a molecular weight of 63.2 kDa. These data suggest that isoforms 1 and 2 are dimers of identical size subunits, and that the basis for their difference is due only to charge. The size of the A-type subunit is identical with that previously observed for human GPI (8) as well as the enzyme from other species (3,13,30,31). In contrast, the two more acidic isozymes (3 and 4) were found to contain a larger subunit (Fig. 3A lanes 4 and 5). The most acidic isozyme (4; pI = 8.62) appeared to be a homodimer of subunits with a molecular weight of 69.8 kDa (designated the B-type subunit). Isozyme 3 with a pI of 8.84 appeared to be a heterodimer composed of both Aand B-type subunits. The isoforms were probed with rabbit antiserum raised against human GPI which had been isolated using fibrous cellulose phosphate. This antiserum was used as the primary antibody and was followed by secondary antibody conjugated to the peroxidase ELISA system. As shown in Fig. 3B, the immunochemical reaction was

HUMAN

GLUCOSE-B-PHOSPHATE TABLE

ISOMERASE

123

ISOFORMS

I

Comparison of Purification of GPI Using Fibrous and Cross-Linked Spherical Particle Phosphocellulose” Total activity (units)

Procedure Dialysate PC: cross-linked PC: fibrous ’ For experimental

Total protein btz)

209 115 87

Specific activity (units/mg)

1390 0.14 0.13

details see Fig. 1 and Experimental

3

4

5

0.15 821 669

(1) 5470 4460

Yield (%)

Time required (h)

(100) 55.0 41.6

4.8 30.8

Procedures.

complete only with the isozymes containing the A-type subunit (i.e. isozymes l-3). No immunochemical reaction was observed with the homodimer containing the B-type subunit. The two types of GPI subunits could be preparatively isolated by SDS-PAGE coupled with extraction of the gel, or by electrophoretic elution (32, 33). The first method was slower and resulted in yields of 60% or less. The electrophoretic method gave higher recoveries and less contamination by gel-derived material. In this work, preparative SDS-PAGE with continuous elution was used to isolate the GPI subunits. Fig. 4 shows that the two GPI subunits could be isolated successfully by preparative SDS-PAGE, with at least 90% recovery of the protein as determined by Bradford assay. Utilizing the above methods it was thus possible to fully isolate each of the four GPI isoforms as well as each of the two types of subunits. The physical, chemical, and catalytic properties of the GPI isoforms are summarized in Table II.

12

Purification (fold)

6

Catalytic and stabilityproperties of the isoforms. It is of particular note that the specific activity of the isoforms containing the B-type subunit is much lower than the isoforms containing the A-type subunit. The activity of isozyme 4 (B-type homodimer) is only about 10% that of the native A-type homodimer. The AB-type heterodimer exhibits an intermediate level of activity, approximately the mean value of the two homodimers. In addition, isoforms 1 and 2 containing the A-type subunit are more stable than the isozymes 3 and 4 containing the Btype subunit at alkaline pH (Table II). Chemical characterization of the subunits. Table III summarizes the results of amino acid analyses of the two subunits. Amino acid analysis of A-type subunit gave results essentially identical to the composition previously reported for the human enzyme (8,10,34) and showed a high degree of similarity to the enzyme from other species (3, 11, 31). The amino acid analysis of the B-type subunit presented a significant difference from the Atype subunit and GPI from other species, especially in the content of Ala, Gly, Ile, and Ser. The Ala, Gly, Ile, and Ser contents of the B-type subunit are substantially higher than those of the A-type subunit, while the Thr

0

B

A &I,

FIG. 2. Electrophoretic patterns of GPI isoforms from human placenta, Specific GPI activity stains were carried out after isoelectric focusing (pH 8.0-10.5) as described under Experimental Procedures. Lanes (1) and (2), GPI purified by cross-linked spherical particule phosphocellulose; lanes (3) to (6), GPI isozymes separated by preparative isoelectric focusing: (3), GPI isozyme 1; (4), GPI isozyme 2; (5), GPI isozyme 3; and (6), GPI isozyme 4.

1234

5

1

2

3

4

5

FIG. 3. SDS-PAGE of the GPI isoforms. Cross-linked spherical particulate phosphocellulose-purified GPI (lane 1) and IEF-separated GPI isozymes l-4 (lanes 2-5) were subjected to SDS-PAGE. Coomassie blue stained gel (A) and an immunochemically stained corresponding Western blot (B) are shown. Experimental details are given under Experimental Procedures; lane 2, GPI isozyme 1; lane 3, GPI isozyme 2; lane 4, GPI isozyme 3; lane 5, GPI isozyme 4.

124

SUN

ET AL.

TABLE Properties Relative Isoform 1

2 3 4

amountb (So)

9.13 9.00

44-50 35-37

8.84 8.62

10-15

of Human

GPI Isoforms

Subunit MW’ &Da)

Subunit type’

63.2 63.2 63.2 + 69.8

A A A+B B

l-5

II

69.8

Specific activityd (units/mg)

Relative N-terminus

1099

Blocked Blocked Blocked Blocked

1003 549 115

stability’ (So) 93

88 81 81

a The pZ was determined by isoelectric focusing on polyacrylamide gels against IEF standards. b Represents the relative amount of protein (%) determined by densitometric quantitation of silver-stained nondenaturing gels as described under Experimental Procedures, except the resolving portion contained an S-25% gradient in acrylamide. ’ Determined by SDS-PAGE (10%) against molecular weight standards (12 to 97 kDa). d The isozymes were resolved by preparative IEF and specific activities (units/mg) determined spectrophotometrically. e Relative stability (%) was determined as the enzyme activity remaining after incubation of the isoforms (0.5-2 pg/ml) for 40 min at pH 10 in 10 mM Caps buffer containing 5 mM BME and 5 mM EDTA. The activity of each of the isoforms at t = 0 is taken as 100% to correct for the differences in their specific activities.

and Leu contents of the B-type subunit are lower than those of the A-type subunit. The amino terminal analyses of the A- and B-type subunits resulted in no identifiable amino terminal residue. Therefore, it appeared that the amino termini of both subunits were blocked as previously observed. for the enzyme from human (lo), bovine (ll), and rabbit (31). As a means of comparing the primary structure of the two subunits, peptide mapping after CNBr treatment was performed. Amino acid analyses showed the presence of 12 or 13 methionine residues per subunit. Pep-

tide mapping by SDS-PAGE after CNBr treatment indicated approximately 17 and 15 peptides for the A- and B-type subunits, respectively (Fig. 5). The absence of bands in the 63-69 kDa region corresponding to intact subunits indicate that there was no undigested core. It is also evident that the banding patterns for the two subunits, i.e., the number and relative intensities of the bands as well as the molecular weights of the peptides, are different. The peptide fragmentation data suggest

TABLE

III

Amino Acid Composition of GPI Subunits

1

z iii 6 5

Amino acid Asx Thr Ser Glx GUY Ala Val Met Ile Leu

Tyr 0.000 0

10

20

30

FRACTION

40

50

60

70

NUMBER

FIG. 4. Preparative isolation of GPI subunits by SDS-PAGE. Samples were electrophoresed in tube gels (l-cm diameter, stacking gel, 1 cm, 3% acrylamide; resolving, gel 4 cm, 10% acrylamide) at 8 mA and eluted at 10 ml/h flow rate. Fractions (0.75 ml) were collected after the tracking dye eluted. The protein concentration was determined by measuring absorbance at 280 nm. The insert shows SDS-PAGE of isolated human GPI subunits; left, GPI purified by phosphocellulose; center, isolated A-type subunit; right, isolated B-type subunit.

Phe LYS His Arg Pro CYS Trp

GPI”

Subunit Ab

Subunit B”

NLKd

58 38 31 65 48 40 32 12

61.5 f 2.3 37.8 + 0.8 30.0 f 0.8 66.8 + 1.8 45.0 + 2.2 47.3 f 2.1 35.3 -c 1.5 13.5 f 1.1 30.5 +_ 1.5 58.5 f 1.5 15.0 + 0.7

61.3 + 0.5 31.3 f 0.5 37.0 + 0.8 71.3 f 1.7 66.3 + 0.9 80.0 + 1.6 38.0 f 0.8 12.3 312.5 38.7 f 4.5 51.0 f 2.4 18.3 + 1.2 28.3 2 1.2 41.6 f 1.7

55 35 35 60 40 42

29 59

14 28 39

24 29 ND’

3 11

29.5 f 1.1

37.3 2 2.3 21.5 + 0.9 28.3 f 1.1 ND ND ND

’ From Lu et al. (16). * Results are from four determinations. ’ Results are from three determinations. d Calculated from the pig GPI cDNA (3). e ND not determined.

19.7 * 1.2 29.0 3~3.2 ND ND ND

29

14 34 55 13 26 38 20 20 23 4 13

HUMAN

residue 1

GLUCOSE-B-PHOSPHATE

Mr

WDa)

ISOMERASE

ISOFORMS

5

125

Mr (kD Ia) -94.0 k67.0 -43.0 -30.0

(167-263)

-16.95 -14.40

(l-65,494-5 (438-493)

7 6

!--8.60

(358-401) (264-303) (130-166) (402-437) (85-119,303

4 .84 4 4 1813

-2.51

'-6.21

(339-357) (68-84) (120-129)

FIG. 5. Peptide maps of human GPI subunits after cleavage at methionines. Human GPI A-type subunit (lane 2) and B-type subunit (lane 3) were reacted with CNBr and the peptides generated were analyzed by SDS-PAGE on 20% gels. Lanes 1,4, and 5 contain molecular weight markers. Experimental details are given under Experimental Procedures. Molecular weight markers are: rabbit muscle phosphorylase b. 94.0 kDa; BSA, 67.0 kDa; egg white ovalbumin, 43.0 kDa; bovine erythrocyte carbonic anhydrase, 30.0 kDa; soybean trypsin inhibitor, 20.1 kDa; bovine milk a-lactalbumin, 14.4 kDa; myoglobin polypeptide backbone-intact, 16.95; fragment I + II, 14.4; fragment I, 8.16; fragment II, 6.21; fragment III, 2.51 kDa.

that the two subunits are structurally different and the B-type subunit does not represent a mere terminal extension. If the two subunits differed only in terminal extensions, one would expect most of the internal peptides to match, even with the single difference in methionine content. Since the differences in the subunit properties could be due to different degrees of glycosylation, considerable effort was extended to evaluate the carbohydrate content of each of the two types of subunits. Figure 6 shows the results of PAS staining of the GPI isoforms for carbohydrate content (A) and Coomassie blue staining for the total protein (B). Fetuin, DNase I, RNase B, and ovalbumin were used as positive controls while bovine serum albumin and the other molecular weight marker proteins (lane 12) served as negative controls (i.e., lane 12 contains both positive and negative controls). Fetuin has both O-linked and N-linked carbohydrate moieties (35) while RNase B, DNase I, and ovalbumin contain only single N-linked oligosaccharide chains. All of the positive controls produced an intense purple color on the gel (lanes 1, 2, 3, 9, and 12) indicative of the carbohydrate, while the negative controls did not react (lanes 11 and 12). Figure 6 (lanes 5 to 8) also shows that GPI from all species examined (human, rabbit, bovine, and yeast)

failed to yield positive PAS reactions. There was no difference in the PAS sensitivity of the two types of human subunits (lane 7). In spite of these results it was deemed important to verify by an independent method that the isoforms of GPI are not due to differential glycosylation (see Discussion). Thus, samples were treated with PNGase F, which has broad specificity for hydrolysis of high mannose type and complex type asparaginelinked oligosaccharides (24, 36). After PNGase F treatment, glycoproteins typically exhibit altered mobility on SDS-PAGE (24). As shown in Fig. 6 (lanes 4 and lo), DNase I and RNase B no longer reacted with the Schiff s reagent and their relative mobilities on SDS-PAGE were altered (Fig. 7, lanes 4 vs. 5, and 14 vs. 15). Fetuin still stained positive with the Schiff’s reagent but its relative mobility was altered (Fig. 6, lanes 1 and 2; Fig. 7, lanes 1 and 2). This is due to the presence of both Nand O-linked carbohydrate groups on fetuin, of which PNGase F can only remove the former. Neither the two types of subunits from human GPI nor the GPI from the different species examined exhibited any detectable change in mobility after PNGase F treatment (Fig. 7, lanes 5 to 12). As a further test, after PNGase F treatment aliquots containing 0.5 mg of fetuin, DNase I (positive control), BSA (negative control), or GPI samples

126

SUN

1234

ET AL.

5

6

7

8

9101112

Mr

CkDa)

-94.0 fetuin

-

RNase

B -

-67.0

-20.0

1234

5

6

7 as-.

8

9101112

Mr

(kDa)

-94 .o fetuin

-

ovalbumin

-

DNase

I -

-67.0 -43.0

-30.0

RNase

B -

FIG. 6. Carbohydrate and protein staining on SDS-polyacrylamide gels. (A) PAS-stain, after l-h incubation in Schiffs reagent in darkness, and (B) Coomassie blue double stained the gel as described under Experimental Procedures. Lanes 1 and 2, fetuin; 3 and 4, DNase I; 5, rabbit GPI; 6, yeast GPI; 7, human GPI; 8, bovine GPI; 9 and 10, RNase B; 11, BSA; 12, molecular weight standards (rabbit muscle phosphorylase b, 94 kDa; BSA, 67 kDa; egg white ovalbumin, 43 kDa; bovine erythrocyte carbonic anhydrase, 30 kDa; soybean trypsin inhibitor, 20.1 kDa; bovine milk ol-lactalbumin, 14.4 kDa). Samples in lanes 2,4, and 10 were incubated with PNGase F before electrophoresis.

were removed and deproteinated and the supernatant solutions analyzed by HPLC using pulsed amperometric detection for mono- and oligosaccharides (25, 26). The release of carbohydrate from positive controls was detected, but there was no release of carbohydrate from any of the GPI isoforms or the negative controls. DISCUSSION

Isoforms of GPI have been recognized in various species for over 20 years. Yet, in most cases, structural analyses have shown that the multiple forms are due to modifications (e.g., oxidation or proteolysis) occurring in cell extracts or during the purification procedures. An exception is the accumulation of specific deamidated isoforms of GPI in aging cells and tissues (13). The deamidated

isoforms appear to be due to specific Asn-Gly sequences (11). In the present study isozyme 2 most likely represents a deamidated form of isozyme 1, as is the case for bovine GPI. The question arises, “why have the other more acidic isoforms containing the B-type subunit (isozymes 3 and 4) not been previously observed?” Our first concern was that these isoforms might be artifacts, and a systematic study was undertaken to evaluate this possibility. The source of the enzyme was considered, and numerous isolations were conducted from different placentae (over 25 different placentae have been utilized to date). Identical results were obtained in all cases, and thus the isoforms are not due to allelic heterogeneity. Placentae immediately processed at the time of delivery showed no difference with those which had been frozen prior to the isolation process. Isolations conducted in the

HUMAN

1234

5

6

7

6

9

IQ

GLUCOSE-6-PHOSPHATE

Ii

12 13 14 15 16

MI

-20.1

-14.4

FIG. after bered F for rabbit GPI;

7. Comparison of the relative mobilities of proteins before and PGNase F treatment. Proteins were analyzed before (odd numlanes) or after (even numbered lanes) incubation with PNGase 18 h at 37°C. Lanes 1 and 2, fetuin; 3 and 4, RNase B; 5 and 6, GPI; 7 and 8, yeast GPI; 9 and 10, human GPI; 11 and 12, bovine 13 and 14, DNase I; 15 and 16, BSA.

presence of various concentrations of reducing (15-60 mM BME) and chelating (0.34-1.0 mM EDTA) agents gave identical results. The answer to the question of why the two isoforms containing the B-type subunit have not been previously recognized appears to be related to the differences in the two types of cellulose phosphate affinity ligands used in the isolation processes. In the past GPI was isolated using fibrous cellulose phosphate, which required much longer chromatographic times and resulted in lower yields. In view of the relative instability of the isoforms containing the B-type subunit and the low abundance of the B-type subunit (approximately lo%), the failure of the previous isolation procedures to yield detectable levels of the isoforms containing the Btype subunit is not surprising. Fazi et al. (37) recently reported the existence of an age-dependent isoform of GPI from human erythrocytes. This isoform resembles the forms we observe in placenta in that they (a) exhibit lower catalytic activity, (b) have markedly lower immunological cross-reactivity, and (c) are distinctly different from the deamidated forms. The isoform from erythrocytes differs from the isoforms we observe only with regard to stability. Unfortunately, the aging-related erythrocyte isoform was not isolated, its molecular weight determined, or otherwise characterized. Structural studies on the A- and B-type subunits reveal several important differences. First, the B-type subunit is larger than the A-type subunit. The initial hypothesis considered was that the B-type subunit might be glycosylated. While a cytosolic protein is not expected

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127

to be glycosylated, this hypothesis was attractive since (a) it could account for the apparent changes in migration on SDS-PAGE, (b) several potential glycosylation sites (Asn-X-Ser/Thr) appear from the sequence data on GPI (3, 38, 39) and NLK (l), and (c) NLK has an extracellular function. This possibility was examined in detail and, to our knowledge, the studies reported here are the first direct analyses designed to determine if GPI is glycosylated. In all cases (i.e., the A- and B-type subunits of human GPI as well as GPI from different species) there was no indication of any carbohydrate bound to the protein. Another approach was to examine the primary structures of the two subunits. Amino terminal analyses proved to be inconclusive because the amino terminus of GPI is acetylated (40). Amino acid composition analyses of the A-type subunit were essentially indistinguishable from published data. On the other hand, the data from the B-type subunit showed significant differences. In addition to what might be considered conservative substitutions of serine for threonine and isoleucine for leucine, the B-subunit exhibits significantly higher levels of glytine and alanine. The possibility of these differences arising from contaminants in the buffers or materials used in the purification processes was carefully examined and eliminated. Calculations based on the differences between the A-type and B-type subunit predict that the “extra” portion of the B-type subunit to be approximately 65 amino acids, and composed primarily of glycine (32%) and alanine (51%). This would represent a highly hydrophobic addition to the A-type subunit. Hydrophobic leader sequences or tails are well documented to be involved in transport of both precursors [e.g., reviewed in (41)] and degradation conjugates of proteins, and could be considered as possible explanations for the larger B-type subunit. However, the differences in the peptide maps, as well as the lack of any potential coding sequences [based on the currently available nucleic acid sequences on both GPI (3) and NLK (l)] consistent with such a region of either protein do not favor this possibility. GPI from Trypanosoma brucei is 5 kDa larger than other GPI molecules, which is due to a N-terminal extension (42). This extension appears to be an integral part of GPI and not a cleavable leader sequence, is not present in any other GPI, and its amino acid composition is too diverse to account for the large increase in glycine and alanine observed in this study. Alternate splicing at the intron-exon junctions could explain the differences in size, amino acid composition, and peptide maps, but the lack of genomic sequence data precludes such an analysis at this time. The peptide fragmentation experiments were designed to gain further information regarding the nature of the structural differences between the two types of subunits. Since GPI is a rather large, basic polypeptide

128

SUN

(3, 8, 10, 11, 31) tryptic fragmentation has long proved to be a problem in fingerprinting the enzyme (8). We chose to conduct primary fragmentation at the methionine residues. Random distribution of methionines would result in CNBr peptides of about 5 kDa, and the predicted CNBr fragmentation pattern from the cDNAderived amino acid sequence of pig GPI is shown in Fig. 5. The CNBr fragmentation peptide maps of the two subunits of human GPI (Fig. 5, lanes 2 and 3) show that the fragments from the A-type subunit are in this predicted range while the peptides generated from the Btype subunit are much larger. The difference in the peptide maps, whether due to different methionine locations or due to incomplete hydrolysis of the B-type subunit because of differences in primary, secondary or tertiary structure, provides additional evidence that the two subunit types are distinct. Clues to the origin of the B-type subunit may be obtained from the drastically different catalytic activities of the AB-heterodimer (isoform 3) and the BB-homodimer (isoform 4). Structural studies utilizing site-specific chemical modifications, immobilized dimers and monomers (43), and crystallography have shown that GPI contains two active centers which are located in the subunit-subunit interface (44, 45). Only the dimer is catalytically active and each of the catalytic centers is composed of residues contributed from both subunits. Thus, the proper subunit-subunit fit is critical (43). The Btype subunit could represent a molecule similar to the GPI subunit, with homology capable of forming a dimer but with sufficient differences to prohibit catalytic activity of the B-subunit. This would account for the reduced activity of the AB-heterodimer (isoform 3) and the very low activity of the homodimer of B-type subunits (isoform 4) (Table II). The B-type subunit could have several origins: (I) a precursor molecule with an additional hydrophobic domain (Ala and Gly) which is normally removed to yield the A-type subunit is a possibility but is not favored (vide suprcz); (II) the B-type subunit could be a degradation product (such as an ubiquinated form), but the simple addition of a ubiquitin molecule to the Atype subunit could not account for the differences in the amino acid composition of the two subunit types; (III) the isoforms result from dimerization of the A-type subunit with another protein. Due to high sequence similarity neuroleukin is an obvious candidate. However, from the structural studies presented above, the B-type subunit does not appear to represent mature or incompletely processed NLK. Additional structural analyses of the NLK, the GPI, and the new GPI isoforms reported here, including their tissue distribution, are required to answer these questions.

ET AL. vanced Technology and Research Program (Wound Healing and Aging No. 2147). The authors gratefully acknowledge the technical assistance of Mr. M. Tatarko. We also thank Ms. J. Martin for typing the manuscript.

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