Studies on the structure of human α2-macroglobulin IV. Analysis of the microheterogeneity by isoelectric focusing

371 (1974) 168-176 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

Biochimiea et Biophysica Acta,

BBA 36832 STUDIES ON T H E S T R U C T U R E OF H U M A N ct~-MACROGLOBULIN IV. ANALYSIS FOCUSING

OF T H E

MICROHETEROGENEITY

BY ISOELECTRIC

JEAN-PIERRE FRt~NOY* and ROLAND BOURRILLON Laboratoire de Bioehimie, Centre de Reeherehes sur les Protdines, Faeultd de MOdecine LariboisiOreSaint-Louis, 45 rue des Saints POres, 75006 Paris (France)

(Received April 17th, 1974)

SUMMARY Four molecular forms were isolated from human a2-macroglobulin by isoelectric focusing in a pH 4-6 gradient. They were found to be homogeneous by immunoelectrophoresis and polyacrylamide gel electrophoresis. The isoelectric points of the four peaks were 4.97 ± 0.02, 5.12 ± 0.02, 5.30 ± 0.02 (main component) and 5.50 ± 0.02, respectively. The amino acid composition of the fractions did not differ significantly, whereas a lowering of the sialic acid, and an increasing of the mannose/galactose ratio were noticeable as the pI of the fractions increased. Comparable patterns were observed with the isoelectric focusing of the individual and pooled preparations of a~-macroglobulins. The data suggest that the microheterogeneity is related to a more or less complete glycosylation which was confirmed by the isoelectric fccusing of neuraminidasetreated ct2-macroglobulin.

INTRODUCTION ct2-Macroglobulin is one of the main plasma glycoprcteins [1]. It binds many proteinases without blocking their active centers. Several investigators, using various separation methcds, have demonstrated the microheterogeneity of human a2-macroglobtdin [2, 3]. Saunders et al. have pointed out that this microheterogeneity is probably due to the interaction of cJ2macroglobulin with proteases [3]. The heterogeneity of the a2-macroglobulin glycoFeptides prepared by pronase proteolysis, was demonstrated by chromatography on DEAE-cellulose [4] and by zone electrophoresis [5]. Glycopeptide analysis showed a high degree of variation in size and composition of the oligosaccharide units. It has been suggested that the structure of the carbohydrate units of human a2-macroglobulin represent varying * Attach6 de Recherche au C.N.R.S.

169 stages of completion on a single type of structural pattern. Each carbohydrate unit appears to contain an internal portion composed of three mannose and two N-acetylglucosamine residues to which the more external sugars are bound in variable amounts [4]. This present paper reports the use of isoelectric focusing to separate the different molecular forms of human a2-macroglobulin. The isolated forms have been characterized and the molecular heterogeneity related to the microheterogeneity of the carbohydrate units. MATERIAL AND METHODS

Human a2-macroglobulin. Human a2-macroglobulin was prepared from pooled serum, by (NH4)2SO4 precipitation, Porath column electrophoresis and Sephadex G-200 gel filtration [6]. The homogeneity of the preparation was checked by analytical ultracentrifugation, polyacrylamide gel electrophoresis at pH 7.5 and 10.2, and immunoelectrophoresis. Individual human a2-macroglobulins were purified as previously described [7], with an additional gel filtration step on Sephadex G-200 [8]. One sample of azmacroglobulin prepared by rivanol precipitation [9] was kindly given by J. Bieth (INSERM, Strasbourg). Treatment with neuraminidase. A sufficient amount of neuraminidase (Sigma, type VI, chromatographically purified from CIostridium perfringens) was used to give a ratio of 30 munits of enzyme/rag of a2-macroglobulin. The enzymic treatment was performed at 37 °C in 0.1 M sodium acetate buffer, pH 5.0 with NaN 3 as the bacteriostatic agent. The released sialic acid was assayed on aliqucts by the thiobarbituric acid assay [10]. After 24 h, the sialic acid was removed by extensive dialysis against the same buffer. The protein-enzyme mixture was reincubated for 16 h at 37 °C and then dialyzed at 4 °C against distilled water. Isoelectric focusing. Separations by isoelectric focusing methods were carried out according to Vesterberg and Svensson [11], with the LKB Model 8102 jacketted column (440-ml capacity). The pH gradient of the 1 ~ carrier ampholytes (ampholines) was pH 4-6 in a linear sucrose gradient (0-47 ~o) or pH 4-7 (0.75 ~ ampholines 4-6, 0.75 ~ ampholines 5-7) in the same sucrose gradient. In all experiments, the bottom electrode solution contained 55 ~o (w/v) sucrose and 1 ~ H2SO4 and the top electrode solution contained 2 ~ ethylenediamine. Electrophoresis was run at 18 °C at a voltage of 800 V for 48 or 72 h. The content of the column was then emptied by gravity at a flow rate of 100 ml/h. The pH of each fraction was measured immediately after collection with a Radiometer 25 pH meter. Re-isoelectric focusing experiments were performed in analytical polyacrylamide gels, with a pH 4--7 gradient, at 4 °C, according to the method of Doerr and Chrambach [12]. Following the electrophoresis the gels were fixed in trichloroacetic acid and then stained with Coomassie Brilliant Blue G-250 as described by Radola [13] Gelfiltration. After pooling and concentration by dialysis under vacuum the fractions obtained by isoelectric focusing were cleared from ampholines and sucrose by filtration through a Sephadex G-25 column (21 cm × 2 cm) in 0.1 M pyridine acetate buffer, pH 6.4 at 4 °C. DEAE-cellulose. DEAE-cellulose (DE 52 Whatman) was equilibrated with

170 0.02 M phosphate buffer, pH 7.4 and poured into a column (11 cm × 1.5 cm). The column was eluted by a linear salt gradient from 0 to 0.2 M NaC1, in 0.02 M phosphate buffer, pH 7.4. The flow rate was approximately 6 ml/h and 2-ml fractions were collected. Polyacrylamide gel electrophoresis. Conditions for polyacrylamide gel electrophoresis were described by Rodbard and Chrambach [14]. Photopolymerization and electrophoresis were performed at 0 °C. The multiphasic buffer system B (Tris-HClphosphate-glycinate) operative at pH 10.2 (0 °C) was used. The separation gels were 5.88 ~ acrylamide and 0.12 ~ methylenebisacrylamide. After electrophoresis, the gels were stained for protein with Coomassie blue. Immunoelectrophoresis. Immunoelectrophoresis was carried out according to Hirschfeld [15]. Human plasma protein antiserum from horse was purchased from the Pasteur Institute (No. 907). Rabbit monospecific antiserum to human a2-macroglobulin was prepared by the injection of 0.5 ml of a 1:1 emulsion of 1 mg a2-macroglobulin [6] and complete Freund's adjuvant into each hind foot pad. Four injections were given at intervals of 8 days with exsanguination 21 days after the last injection. Chemical analyses. For the amino acid analyses, 100 nmoles of protein were hydrolysed, under vacuum with 6 M HC1, for 16 h at 105 °C. The hydrolysates were analysed on a Beckman Unichrom amino acid analyzer according to D6v6nyi [16]. The carbohydrates were analysed by gas-liquid chromatography according to the technique of Chambers and Clamp [17]. The results are the average of two or four determinations. RESULTS

Isoelectric focusing of pooled human a2-macroglobulin The results of the isoelectric focusing of %-macroglobulin isolated from human 2.0

/ /f 1.5

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4

0.5

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ioo FRACTION

NOMSEn

Fig. 1. Isoelectricfocusingabsorbance-pH pattern of 50 mg of human a~-macroglobulinin a pH 4-6 gradient. 3.8-ml fractionswere collected.

171

Fig. 2. (a) Polyacrylamide gel electrophoregram, pH 10.2, 0 °C, of isoelectric focusing fractions. (b) Immunoelectrophoregram of isoelectric focusing fractions. AH, human plasma protein antiserum; A a~-MG, human a~-macroglobulin antiserum.

serum are shown in Fig. 1. Four major peaks with a few small peaks were isolated. The isoelectric points of four peaks A, B, C, D were determined to be 4.97 ± 0.02, 5.12 ± 0.02, 5.30 ± 0.02 (main component) and 5.50 ± 0.02, respectively. The contents of the tubes corresponding to each fraction were pooled, concentrated by ultrafiltration and the salts were removed by filtration on Sephadex G-25. Polyacrylamide gel electrophoresis of the four peaks (Fig. 2a) shows only a~-macroglobulin always associated with a faint band already described [6, 18]. Furthermore, the four fractions contained only a2-macroglobulin as studied by immunoelectrophoresis against total plasma protein antiserum from horse (Fig. 2b). In order to test the isoelectric homogeneity of the different molecular forms of human a2-macroglobulin, isolated fractions were re-isoelectric focused in polyacrylamide gel, within the pH range of 4-7. In each case a single and sharp protein band was observed after staining. TABLE I CARBOHYDRATE COMPOSITION OF F O U R FRACTIONS OF ISOELECTRIC FOCUSING OF H U M A N %-MACROGLOBULIN Values expressed as g per 100 g protein. A

A

B

C

D

Mannose Galactose N-Acctylglucosamine N-Acctylneuraminic acid

3.04 2.79 2.74

3.14 1.72 3.20 2.33

3.01 1.45 3.39 2.23

2.99 1.28 3.36 1.62

Mannose/Galactose

1.09

1.83

2.08

2.34

3.57

172

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,¢( I

0.5

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0.2

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0.1

J 50

!

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100

150

Fig. 3. DEAE-cellulose chromatography of 60 mg of human a~-macroglobulin. The column was eluted with starting buffer followed by a gradient of 0-0.2 M NaCl (200 ml), in the same buffer. 2-ml fractions were collected. The amino acid composition o f fractions A, B, C, D does not present significant differences, whereas the carbohydrate composition exhibits discrepancies (Table I). A lowering o f the sialic acid content was ascertained as the pl of the fractions increased. The mannose/galactose ratio increases similarly at the same time as the pI. DEAE-cellulose c h r o m a t o g r a p h y with a linear salt gradient was unable to separate the different molecular forms o f a2-macroglobulin (Fig. 3). Isoelectric focusing in a p H 4-6 gradient of a2-macroglobulin eluted f r o m the DEAE-cellulose column 2.0 •

5

1.5

5

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1.0

/ 4

0.5

3

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Fig. 4. Isoelectric focusing absorbance-pH pattern of 50 mg of human a2-macroglobulin after DEAE-cellulose chromatography. Carrier ampholytes covering a pH range between 4 and 6 were used and 3.8-ml fractions were collected.

173 TABLE II ISOELECTRIC F O C U S I N G OF H U M A N a.2-MACROGLOBULINS Protein

Isoelectric points*

Native Pooled serum Pooled (rivanol preparation) Individual male Individual female Modified After DEAE-cellulose chromatography Neuraminidase treated

4.97, 4.82, 4.96, 4.87,

5.12, 5.26, 5.18, 5.33,

5.30, 5.50, 5.48, 5.32, 5.43, 5.59,

4.90, 5.45, (Range 5.0-5.80) 5.87,

* The underlined values are the pl values of main components.

(Fig. 4) showed many components, with a shift towards the more elevated pI (Table II).

Isoelectricfocusing of several individualpreparations of human a~-macroglobulin The heterogeneity of human a2-macroglobulin, as revealed by isoelectric focusing, has also been noted with other preparations, a2-Macroglobulin prepared using the rivanol precipitation technique shows the main pI at 5.26 and the minor pI at 4.82 and 5.49. Two purified proteins from individual human serum, male and female (Fig. 5) showed the pI to be near 5.30 and the minor pI approaching 4.90 and 5.50 (Table II).

Isoelectricfocusing of desialylated a2-macroglobulin The first neuraminidase treatment released about 75 ~ of total sialic acid from human a2-macroglobulin during a 24-h hydrolysis (Fig. 6). After complete enzymic

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,,/

f/ l 0.2

4

0.1

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Fig. 5. Isoelectric focusing absorbance-pH pattern of 18 mg of human female a2-macroglobulin in a pH 4-6 gradient. 3.8-ml fractions were collected.

174

100 z ~

7...

z m

25

i

i 2

i 4

i 8

TIME (HOURS)

i 24

Fig. 6. Neuraminidase treatment of human a2-macroglobulin during the first 24-h hydrolysis. Released sialic acid (NANA) was assayed on aliquots by the thiobarbituric acid assay. 2.0

1.5

|

J l.0

4

i 0.5

,~o FRACTION NURSER

Fig. 7. Isoelectric focusing absorbance-pH pattern of 60 mg of human e=-macroglobulin after complete neuraminidase treatment. Carrier ampholytes covering a pl-t range between 4 and 7 were used and 3.8-ml fractions were collected. treatment no sialic acid was observed by gas-liquid c h r o m a t o g r a p h y analyses. Isoelectric focusing in a p H 4-7 gradient (Fig. 7) showed that this treated material was shifted to a position corresponding to a higher isoelectric point than that of untreated material, pI 5.87 (Table II). F u r t h e r m o r e the protein material was eluted in a single, symmetric and very sharp peak. DISCUSSION A m o n g the multiple molecular forms o f h u m a n a z-macroglobulin in isoelectric focusing, the pI 5.30 o f the major peak agrees with the recent results of H a m b e r g

175 et al. [19]. It is slightly lower than the pI 5.4 as determined by other methods [18, 20]. The pI 5.30 is somewhat higher than those found for animal a-macroglobulins [21,22] some of which, rabbit al-macroglobulin [23], and mouse a2-macroglobulin [24], are claimed to be heterogeneous in isoelectric focusing. The minor peaks of human a2-macroglobulin have never been prepared, though Jones et al. [25] have noticed from one to three thint bands between 5.4 and 5.6 near the major 5.4 component in isoelectric focusing in the polyacrylamide gel. It must be noted that these different molecular forms were not separable by chromatography on DEAE-cellulose, perhaps on account of a desialylating process. This was suggested by the following isoelectric focusing experiment exhibiting a shift towards high pI values. The observed microheterogeneity could be due to different origins [26]. It is thought that it was not due to a chemical modification during the isolation process, for it was performed quickly at low temperature and the DEAE-cellulose step, which may release some sialic acid, was avoided. Ganrot and Laurell [2] by using antigen-antibody crossed electrophoresis discriminated two peaks for az-macroglobulin in human serum. Treatment of the serum with pneumococcal neuraminidase did not cause any obvious change in the degree of electrophoretic heterogeneity or in the mean mobility while the mobility for other plasma proteins is reduced in the typical way. So it was claimed that the heterogeneity depended on the formation of complexes between a2-macroglobulin and other compounds i.e. proteases and hormones. More recently Saunders et al. [3] observed two bands of a2-macroglobulin when polyacrylamide gel electrophoresis was performed with a pH 8.9 gel. When a pH 7.8 gel was used, five electrophoretic species were observed. In both cases, the addition of stoichiometric amounts of trypsin to protein resulted in the disappearance of slower bands leaving only one band on the polyacrylamide gel electrophoresis pattern. So the fast moving component was the az-macroglobulin-trypsin complex and the slower moving material was unbound a2-macroglobulin. The above data suggest that the microheterogeneity of a2-macroglobulin is related to the more or less complete glycosylation of its carbohydrate units. Microheterogeneity was worked out by isoelectric focusing and confirmed by the variation in sialic acid and galactose content. These results agree with the observed heterogeneity for a2-macroglobulin glycopeptides [4, 5]. It is also likely that as-macroglobulin has a slight heterogeneity arising either from a random assortment of possible different subunits [27], or from genetic determination: there are at least eight main antigenic determinants for human as-macroglobulin [28] and allotypes have been described in the case of animal as-macroglobulins [29]. ACKNOWLEDGEMENTS The technical assistance of Mrs Catherine Michon with the amino acid analyses is gratefully acknowledged. This work was supported by grants from the C.N.R.S. (E.R.A. 321), the Facult6 de M6decine Lariboisi~re-Saint Louis, and the I.N.S.E.R.M. (contrat No. 71.1.056.2).

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