Characterisation of an allergen extracted from ascaris suum

lmmunochemistry, 1973, Vol. 10, pp. 815420.

Pergamon Press.

Printed in Great Britain

CHARACTERISATION OF AN ALLERGEN F R O M ASCARIS SUUM.

EXTRACTED

DETERMINATION OF THE MOLECULAR WEIGHT, ISOELECTRIC POINT, AMINO ACID AND CARBOHYDRATE CONTENT OF THE NATIVE ALLERGEN J. A M B L E R , J. N. M I L L E R , P. J O H N S O N and T. S. C. O R R Fisons Ltd., Pharmaceutical Division, Research and Development, Bakewell Road, Loughborough, Leics., England, Department of Chemistry, University of Technology, Loughborough, Leics., England and the Laboratory of Biophysical Chemistry and Colloid Science, Department of Biochemistry, University of Cambridge, Cambridge, England (First received 24 April 1973; in revised form 28 May 1973)

Abstract--An allergen has been purified from whole worm extract of A. suum. The mol. wt has been determined by gel filtration and sedimentation equilibrium to be of the order of 14000, and by amino acid composition to be approximately 12000. The carbohydrate composition of the molecule is apparently extremely low: only 1 per cent glucose and 0.5 per cent hexosamine were detected. lsoelectric focussing of the purified allergen indicated the presence of fractions with isoelectric points of pH 5.0 and 5.2. INTRODUCTION

The ability of certain helminth parasites to e v o k e high titre reagin responses in man and experimental animals demonstrates that these parasites possess an allergenic component. N e m a t o d e allergens have not been fully characterised, h o w e v e r , possibly because pure preparations have been difficult to obtain. N o t only do allergens exist in whole w o r m extracts with an array of tool. wt (Hussain et al., 1972; A m b l e r et al., 1972b) but it has also been shown that allergen may be present with different specificities (Ford, 1971; Barratt, 1972; Ambler et al., 1972b). W e have partially characterised an allergen extracted f r o m another n e m a t o d e N i p p o s t r o n g y l u s brasiliensis and found it to be a glycoprotein with a carbohydrate content of 7.5 per cent. An examination of some of its physicochemical properties showed that in many ways, the allergen bore a striking resemblance to purified allergens f r o m other animal and plant sources. (Ambler et al., 1972a). Because of the small size of N. brasiliensis and the difficulty of obtaining sufficient worms to provide the allergen, we have d e v e l o p e d a biological model w h e r e b y reaginic antisera can be raised in rats against the allergenic c o m p o n e n t s in the whole w o r m extract of A. suum. Difficulties o v e r c o m e by the availability of starting material w h e n working with A. s u u m have been replaced by difficulties in the isolation of a pure allergen fraction. For example it has been shown that at least two allergen fractions A and B can be separated f r o m the whole w o r m extract, and that allergen A can apparently exist in multimolecular forms with regard to mass and charge (Ambler et al., 1972b).

The characterisation of the allergen fraction that is being examined, therefore, is of e x t r e m e importance. EXPERIMENTAL

Materials and methods 1. The preparation o[ Ascaris suum allergen A. (a) The preparation of whole worm extract (WWE) and the separation of allergen A from allergen B by gel filtration through Sephadex G200 and G75 columns have been described in detail previously. (Ambler et al., 1972b). (b) The Sephadex G75 allergen A-rich fractions were dialysed against 0.1M phosphate buffer pH 7.5 and applied to a DEAE-cellulose column at room temperature (bed dimensions 1.5 × 10 cm) equilibrated with the same buffer. A phosphate buffer gradient (0.01M-0.1M) followed by 0.2M phosphate buffer was applied to the column. Biological activities of selected fractions eluted from the column were measured by rat passive cutaneous anaphylaxis (PCA) and by double diffusion in agar gel (Ouchterlony, 1948) using a rabbit anti allergen A precipitating antiserum. (Ambler et al., 1973). (c) The biologically active fractions from the DEAEcellulose column (see results) were pooled, concentrated by Amicon ultrafiltration (UM2 filter, mol. wt retention limit 1000) and dialysed against a CM cellulose column starting buffer (0.01M phosphate pH 5.4). After application of the sample to the column, an elution gradient of 0.01M-0.10M phosphate buffer pH 5.4, followed by 0.20M phosphate buffer pH 5.4 was applied to the column. Allergen A activity in selected fractions was measured as already described. 2. Rat reaginic anti allergen A antiserum (WEP) and passive cutaneous anaphylaxis, have been described in detail previously (Ambler et al., 1972b). 3. Rabbit anti allergen A precipitating antiserum (R. anti A ), Ouchterlony gel diffusion, electrophoresis and ira-

815

816

J. AMBLER, J. N. MILLER, P. JOHNSON and T. S. C. ORR

munoelectrophoresis on Cellogel have been described in detail previously (Ambler et al., 1973). 4. Determination of tool. wt of allergen A by thin layer gel chromatography (TLG). The mol. wt of allergen A was estimated by TLG on 1 mm layers of Sephadex G75 superfine (10x20cm). Cytochrome c (mol. wt 12,000), ribonuclease A (12,800), lactalbumin (14,500), and chymotrypsin (23,500) were used to calibrate the'plates. All reference protein concentrations were 5 mg/ml, and the allergen concentration was 2 mg/ml. Orange dextran was used as void volume marker for the separation (Miller, 1972). 5. Determination of tool. wt by sedimentation equilibrium. The rnol. wt of allergen A was determined in short columns by a low speed equilibrium method, iVan Holde and Baldwin, 1958). The allergen was centrifuged at a concentration of 3 mg/ml in phosphate buffered saline (PBS) pH 7.0, ionic strength 0.1, and at two equilibrium speeds of 14290 and 17980. The mean tool. wt was determined from these two experiments. The initial concentration le,,) in terms of interference fringes was determined in a synthetic boundary cell. The mol. wt M was determined from 2RT M = (f1p_) ¢ o ~

• 2-3(/3dlog e dr ~ .

trifluoracetic acid. After neutralisation, the sample was applied to a chromatography column of type S Chrombeads (Technicon) and eluted from the column with a borate-chloride gradient. The column eluate was analysed for carbohydrate content using a cysteine sulphuric acid reagent, and individual sugars identified by reference to marker standard sugars. 9. Isoelectrie focussing of allergen A. Isoelectric focussing was carried out in a LKB l l 0 m l column in a sucrose density gradient for 48 hr. Two per cent carrier ampholytes (LKB) pH range 3-10 formed the pH gradient. The column was drained through a LKB Uvicord monitor coupled to a linear chart recorder. Two milliliter fractions were collected and the pH was measured immediately using a pH meter. 10. Titration of allergen A. Freeze-dried salt-free allergen was dissolved in 0.06M saline at a concentration of 1 mg/ml. 2 ml of the allergen solution and a saline control were titrated with 0.06M sodium hydroxide or 0-06M hydrochloric acid in a Radiometer autotitrator assembly. Protons released or taken up per molecule of protein (h) were calculated from ]~ (C,,-C,,,,) C,

(I)

A value for t3 was assumed as 0.725. 6. Determination of the sedimentation coefficient. The allergen at concentrations of 3.0. 1-5 and 1.0 mg/ml in phosphate buffered saline (PBS) (I=0.1) pH 7-0 was examined in a cup type synthetic boundary cell at about 20°C and 59780 rev/min in a Beckman model E ultracentrifuge. 7. Amino acid analyses. Amino acid analyses were carried out on duplicate 24- and 72-hr hydrolysates of allergen A using a Jeol SAH amino acid analyser. Tryptophan estimations were made by a colorimetric method (Opienska-Blauth et al., 1963). 8. Carbohydrate analyses on the allergen molecule. Methods for estimating various carbohydrate constituents of nematode allergens have been given previously (Ambler et al., 1972a) and were taken from Kabat and Mayer (1964). Estimations of hexoses, methylpentoses, ketohexoses, pentoses, hexuronic acids, sialic acids and hexosamines were undertaken. In addition 2.20 mg of saltfree lyophilised allergen was hydrolysed with 2M

or

/~ ( C , '

CA)

where C. and C,, = molar concentration of base or acid respectively, C,~ and C,,, = molar concentration of hydrogen or hydroxyl ions and C,,= molar concentration of protein. RESULTS

1. S e p a r a t i o n o / a l l e r g e n A. Allergen A was separated f r o m allergen B as d e s c r i b e d previously. The elution of allergen A from the DEAEcellulose c o l u m n is s h o w n in Fig. 1. Six p r o t e i n peaks w e r e o b s e r v e d and allergen A was f o u n d using the rat P C A test to be c o n c e n t r a t e d in peak 4 (Fig. 1). Small a m o u n t s w e r e d e t e c t e d also in p e a k s 2, 3 and 5. The rabbit a n t i s e r u m R-anti A raised against the c o m b i n e d f r a c t i o n s o f p e a k 4 was f o u n d to give three i n d e p e n d e n t precipitin lines with the antigens in p e a k 4 (Fig. 2). T h e r e was a main arc A, c o r r e s p o n d i n g to allergen A, and t w o i n d e p e n d e n t

304 40-

~

1 2 3

50-

g 60-

70:

G

,

~

.

9o _ 100

.y"

(2)

C,,

i

s

1-30 20

]..

-'~

~ho

....................................... ;,.. ](3 160 200 Elution Volume (ml) Fig. 1. Separation of the allergen A-rich fraction from the Sephadex G75 column chromatography. G denotes the start of the phosphate gradient 0.01M pH 7.5 denotes the addition of 0.2M saline to the final 0.2M phosphate buffer pH 7.5. position of allergen A in the eluate. -..PCA activity.

o_

by DEAE-cellulose to 0.2M pH 7.5. S Shaded area is the

Characterisation of an Allergen Extracted from Ascaris suum--I

817

x

X Y

A

Fig. 2. Ouchterlony plates of the six protein peaks (I-6) eluted from the DEAf-cellulose column against the rabbit antiserum (in centre well). See text for the description of antigens A, X and Y. lines X and Y which were due to other antigens in this preparation. The fractions comprising peak 4 were concentrated by Amicon ultrafiltration, (UM2 filter), and their subsequent elution from the CM cellulose column is shown in Fig. 3, Allergen A activity was detected by PCA only in peaks 3 and 4 eluted with the concentrated phosphate buffer (0-2M pH 5-4) (Fig. 3). Testing all peaks by the rabbit antiserum R-anti A on Ouchterlony plates showed that antigen Y was eluted by starting buffer (0.01M phosphate pH 5.4) and antigen X was eluted in peak 2 (Fig. 4). Neither of the constituents of these peaks had allergenic activity (Fig. 3) and they were not studied further. Peaks 3 and 4 both contained allergen A and were identical antigenically. The fractions comprising peak 3 were concentrated to 0.5 mg/ml and dialysed against PBS before storate at - 20°C. This preparation is referred to as allergen A throughout the work. 2. Determination of mol. wt by TLG. The logarithms of the tool, wt of the reference proteins were plotted against their migratory distances (mm) from the origin line (Fig. 5). The position of allergen A is also shown and corresponds to a mol. wt of approximately 14,000.

3. Determination of mol. wt by sedimentation equilibrium. In equation (1) given earlier

50E o 60-

0.01M pH54

Y

Fig. 4. Double diffusion against the rabbit antiserum (in the centre well) of 1: DEAE fraction 3, 2: CM column fraction 1, 3: DEAE column fraction 3, 4: CM column fraction 2, 5: CM column fraction 2, 6: CM column fraction 4. 100

<

--9~ E E

c 6~

E 80-

o

Lactalbumfn/

8c

Allergen A

m

,=, 70-

RibonucleaseA / Cytochrom~/

60 4"0

4'-1 412 4',3 Logarithm of molecular weight

Fig. 5. Estimation of the molecular weight of allergen A by TLG on Sephadex G-75 [.] Cytochrome c (tool. wt 12,000), ribonuclease A (12,800), lactalbumin (14,500) and chymotypsin (23500) were used as calibrating proteins. d log c/d(r'3 was obtained from the slope of the graph of log,0 c against r e (Fig. 6) where c is the concentration of allergen A in interference fringes and r is the corresponding radial distance. The mean mol. wt was calculated as 14000_+200.

0.1M pH54

3 ~

0.2M pH54

70-

30 ~

80-

m

90

100

,

..............................

f

10 ~

",...............

100

4:4

"

Elution Volume (ml)

"",

200

0

m

Fig. 3. Separation of the allergen A-rich fraction from the DEAE-cellulose column (peak 4) by CM cellulose chromatography. ---PCA activity. % transmission at 280 rim.

818

J. AMBLER, J. N. MILLER, P. JOHNSON and T. S. C. ORR 1"30-

1-20"

1"10Loglo C 1.00.

0.90

0.8C

47 "'

z~8

4'9 5'0 r 2 (cm 2 )

51

Fig. 6. Sedimentation equilibrium of allergen A: A - - A initial speed=21,740 for 2hr; equilibrium after 14,290 rev/min for 20 hr. O----O initial speed = 25,980 for 10 min; equilibrium after 21 hr at 17,980. Allergen at 3 mg/ml in phosphate buffer pH 7.0.

Using a value of 1.85S for s~0~ and the mol. wt value of 14000, fifo was calculated as 1.13. 6. Amino acid analysis. The amino acid analysis results are shown in Table 1. Out of a total of 100-103 amino acids there were 25 acid groups (glutamate, aspartate), five neutral groups (histidine, NH_,-end amino group) and 19 basic groups (lysine and arginine). The mol. wt of the allergen calculated f r o m the mol. wt of the amino acid residues was of the order of 12000. 7. Isoelectric focussing. The results of the isoelectric focussing of allergen A are shown in Fig. 7. T w o protein fractions were observed; a minor fraction at pH 5-0 and a major fraction at pH 5.2. The protein in each fraction was dialysed extensively against PBS pH 7-4 to r e m o v e most of the carrier ampholytes, and the protein concentration was adjusted to 0.4 mg/ml. Both fractions gave the m a x i m u m reaction size of 30 mm with the rat antiserum W E P in the rat PCA. Testing the two fractions against rabbit antiserum (R-anti-A) by immunodiffusion also produced arcs of complete identity.

8. Electrophoresis and immunoelectrophoresis. 4. Determination of the sedimentation coefficient. The allergen m o v e d as a single symmetrical boundary in the centrifugal field at the 3 concentrations of 3-0, 1.5 and 1-0 mg/ml giving c o r r e c t e d sedimentation coefficients of 1.86S, 1.86S and 1.85S respectively, s~0,w was thus taken to be 1.85S.

5. Determination of the frictional ratio (fifo).

Allergen A migrated as a single c o m p a c t zone on electrophoresis at pH 9.24 with the mobility of a bovine serum c~, globulin. I m m u n o e l e c t r o p h o r e s i s with the rabbit antiserum R-anti-A produced one clearly defined precipitin arc. Identical results were produced with the allergen in peak 4 from the CM cellulose column.

Table I. Amino acid analysis of allergen A after 24 hr acid hydrolysis. The results are the means of duplicate estimations Amino acid Tryptophan" Lysine Histidine Arginine Aspartic acid Threonin& Serine b Glutamic acid Proline Glycine Alanine ~_Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine TOTALS

/.tM/mg allergen 0.1385 1.4715 0.4479 0.1098 0.6102 0.3700 0.4150 1.4672 0.1777 0.4955 0.7207 0.1514 0.3194 0.0839 0.2758 0.8342 0.2643 0.1432

Ratio amino acid methionine = 1

Integer

1.47 17.54 5.34 1.31 7.21 4.41 4.95 17.51 ,._ I 5.90 8.59 1.80 3.80 1-00 3.29 9.94 3.15 1.71

1-2 17-18 5 I 7 4-5 5 17-18

100.64

98-103

6 8-9 4 I 3 10 3 2

"Tryptophan was estimated by the method of Opienska-Blauth et al. (1963). ~Hydroxyamino acid data were extrapolated to zero time hydrolysis.

Characterisation of an Allergen Extracted from Ascaris suum--I E

c

70.

.10 .9

g

-8

i 80.

E

0~ .7 z5

pH 5.2

#r~

•6 ~

9o PCA

819

.5

30

"':.. 8

I0

12

14

16

18

20 22 24 26 28 30 32 Fraction Collector Tube Number

34

36

38

40

42

Fig. 7. Isoelectric focussing of allergen A in an ampholyte gradient pH 3-10. - - p r o t e i n concentration (absorbance at 280 nm), A--A pH gradient, ----biological activity by rat PCA. 9. Titration of allergen A. The titration curve obtained for the protein is shown in Fig. 8. 23 acid groups, five neutral groups and 15 basic groups titrated between two pH values of 2.5 and 11.5. 10. Carbohydrate analysis. Only 1% hexose and 0.5% hexosamine were detected in Allergen A. Acid hydrolysis with 2M trifiuoracetic acid and the subsequent chromatographic separation of the sugars confirmed the low content of hexose and identified this as glucose. DISCUSSION

The isolation method for allergen A described in this paper produced an allergen which was apparently homogeneous in the ultracentrifuge and on TLG. Only one precipitation arc was obtained with the rabbit antibody R anti A, and the allergen migrated as a single compact zone on electrophoresis. Isoelectric focussing, however, separated two fractions with isoclectric points of pH 5.0 and 5.2. The small difference might be due to genuine microheterogeneity of the protein or to an artifact caused by binding of ampholytes to the allergen, or 0~

},o.\ 15.

\

Acid Region

, i

~(23groups), ,

Neutral

[ Regfon

i

IAIkaline

~Reg~on

a~"~ 5. ~ ~

i' (5groups) i(15groups

o~ ~

i

O.

~.5.

~"

.a~ 10.

~

15.

~

20.

.

.

t

', ~

-r 25, 2.03.0 4.0 50 6.0 7.0 80 9010011.012.0 pH Fig. 8. Electrometric titration of 2 ml allergen A (1 mg/ml). The reference point of pH 5.45 is that pH which the allergen assumes in 0.06M saline.

by oxidation of certain amino acid residues (Jacobs, 1973). The results obtained indicate that allergen A had a mol. wt close to 14000, and can be regarded as a compact, approximately spherical molecule in view of the low frictional ratio of 1.13. Aggregation of the protein did not occur significantly at concentrations of up to 3 mg/ml as shown by sedimentation velocity studies or at even higher concentrations as shown by sedimentation equilibrium studies. The protein contained a high proportion of charged amino acid residues and a fairly low isoelectric point ( - pH 5.0) which accounted for its high anodal mobility on electrophoresis at pH 9.24. It is difficult to account for the low isoelectric point when there appears to be a balance between the acid and basic amino acid residues. The titration of the protein has clarified this point to some extent. Thus it appears that 4-6 basic groups do not titrate, either because they are buried in the hydrophobic regions of the molecule (although it might be expected that most charged groups would be in the hydrophilic environment close to the surface) or because they might be blocked in some other way (Berrens, 1970). Allergen A contained only 1 per cent hexose as glucose, and 0-5 per cent hexosamine, and thus had a much lower sugar content than N. brasiliensis allergen (Ambler et al., 1972a), though the sugars present were similar. Any discrepancy between the molecular weight obtained experimentally for allergen A, and that calculated from the amino acid residues could not be explained by the sugar content, which would contribute only 400 to the mol. wt if two molecules of sugars were present per allergen molecule. There might be other groups attached to the allergen, therefore, which have not yet been detected. Recently an allergen (ASC-1) containing 11% reducing sugars has been isolated from A. suum by Hussain et al. (1972). These authors determined the molecular weight of their allergen as 30,000-40,000. However they found a smaller tool. wt allergen

820

J. AMBLER, J. N. MILLER, P. JOHNSON and T. S. C. ORR

w h i c h was identical to ASC-1 in its reagin c o m b i n ing p r o p e r t i e s in the rat PCA, but unlike ASC-1, was not i m m u n o g e n i c . H u s s a i n et al. p o s t u l a t e d that ASC-1 c o n t a i n e d a f u n d a m e n t a l allergen unit a t t a c h e d to a specific carrier p r o t e i n w h i c h was n e c e s s a r y for the i m m u n o g e n i c p r o p e r t i e s of the molecule. T h e p r e s e n t a u t h o r s m a d e a similar suggestion a b o u t the same time ( A m b l e r et al., 1972b) a f t e r the d i s c o v e r y that allergen A could exist in w h o l e w o r m e x t r a c t in two mol. wt r a n g e s (15-20,000; 30-40,000). It is likely t h a t allergen A is t h e f u n d a m e n t a l unit w h o s e p r o p e r t i e s are not directly c o m p a r a b l e with t h e p r o p e r t i e s of the ASC-1 of H u s s a i n et al., w h i c h also c o n t a i n s a c a r r i e r protein.

Acknowledgement--We thank Dr. R. Jefferis at the University of Birmingham for carrying out the amino acid analyses.

REFERENCES

Ambler J. and Orr T. S. C. (1972a) Immunochemistry 9, 263. Ambler J., Doe J. E., Gemmell D. K., Roberts J. A, and Orr T. S. C. (1972b) J, Immun. Methods 1, 317. Ambler J., Croft A. R., Doe J. E., Gemmell D. K., Miller J. N. and Orr T. S. C. (1973) J. Immun. Methods 2, 315. Barratt M. E. J. (1972) Immunology 22, 615. Berrens L. (1970) Prog. Allergy 14. Ford G. E. (1971) Immunology 21, 1073. Hussain R., Strejan G. and Campbell D, H. (1972) J. Immun. 109, 638. Jacobs S. (1973) Analyst 98, 25. Kabat E. A. and Mayer M. M. (1964) Experimental Immunochemistry. 2nd Edition. Thomas~ Springfield, Ill. Miller J. N. (1972) J. Chromatog. 74, 355. Opienska-Blauth J., Charezinski, M. and Berbec, H. (1963) Analyt. Biochem. 6, 69. Ouchterlony O. (1058) Prog. Allergy 5, I. Van Holde K. E. and Baldwin R. L. (1958) J. phys. Chem. 62, 734.