Two variants of ragweed allergen Ra3

Two varimHs of ragwdmi

-

a#kqpm Ra3

Lawrence Goodfriend, Ph.D., Marianne Rb, Ph.D., U!f Lmdkuist, Ph.D., and AWul M. Chmdhury, Ph.D. Montreal, Quebec., Canada, und Uppsala. S~cden

Two disc electrophoretic forms of Ru3 (I and II) were isolated by column chromatography in yields uf 30 and 10 mglkg pollen, respectively. On the busis of 87~ carbolrvdrute content (determined for Ru3l and assumed for Ru311), the major und minor jtirms hud essentiull~ identicul molecular weights (12,300 daltons). Although their amino acid compositions were simihr. u number of single residue differences were found. some of which were cor$rmed in separate studies on the complete amino acid sequences of Ru3l und R&Il. In limited trssuvs. no d@rences were observed in their untigenic and ullergenic spec[/icities,

Following

the initial

report’

on the isolation

and

properties of short ragweed pollen allergen Ra3, studies in several laboratories have established this low-molecular weight glycoptotein as an important incntant in ragweed pollinosis2~ 3 and a potentially useful reagent for structure-activity4 and immunogenetic studies.3* *’ In further work with the allergen in this laboratory,” we consistently observed the presence of major and minor disc electrophoretic components in preqarations of the allergen isolated from different lots of short ragweed pollen obtained from the same (commercial) source as in the initial study.’ Surprisingty, the components could be resolved to disc electrophoretic homogeneity by multiple cycles through Sephadex G-50 superfine. Chemical and immunofogic comparisons, though limited, suggest that the major and minor components constitute structurally closely related isoantigenic and isoallergenic forms, termed Ra3I and Ra3II. MArEmALsAuDMEl?#oDs Short ragweedpollen was purchasedfrom Sharp & Sharp (Everett, Wash.). Column chromatographic and auxiliary conditions for isolation lulose (DEAE)-fraction

of Ra3 from diethylaminoethyl celC of water-soluble ragweed (WSR)

-..__ From the Division of Clinical Immunology, Royal Victoria Hospitr, Montreal, and the Dqartmcnt of Biochemistry, Pharrnacia I2iqnoh.s AB, Uppsala. Supported in pati by a grant (MT 2010) from the Medical Research Council , Canada. Received for publication June 30, 1980. Accqteci for publication Dec. 18, 1980. Repint requests to: Dr. L. Goodfriend, Division of Clinical Immunnlogy, Royal Victoria Hospital, 687 Pine West, Montreal. Quebec. Canada H3A IAI. 0041-0749181/040299+06$oo.6o10

G 1981 The C. V. Mosby

with minor modifications (see Mow). Yields of the antigen (and its variants) were based

were as described previously’

on an assumed Ef ,“,!$,, “,,, = I. I I. Disc electmphoresis was performed at pH 4.3 in 15% polyacrylamide gels by the methodof Reisfeld et al .I’ Bands were fixed and stained according to Koening et aI.” Molecular weights were determined as detailed elsewhere” in a Beckman Model E uItmcen@ifuge (equipped with ultraviolet optics, scanner, and recorder) by the overspeedmodification of the conventional sedimentation equilibrium technique.* Partial specific volumes were catculated from the (molar) amino acid compo&ions’* and correctediS for total carbohydrate content. The latter was determined spectrophotometrically by the ttyptophan reaction” using arabinose as standard. Amino acid compositions of Ra31 and Ra311 were determined as described previously’ in a Beckman Analyzer Model 12OBon duplicate aliquots of native (unoxidized) protein. Tryptophan was determined spectmphotometrically,‘5 assuming 4 tyrosine residues per mole Ra3I. Ra3II as established by sequence analysis.t The number (N) of amino residues was calculated from the following equation: N = FM amino acid/FM sample, assuming 11,000 d&tons for the molecular weight of the protein moieties of Ra31 and Ra311. Immunodiffusion tests were done in agar using rabbit antiserum to crude aqueous extract (ReMh Resources Branch, National Institutes of HeaIth) and to Ra31. The latter was prepared by four weekly injections intramuscularly of 0.5 mg Ra3I in Freund’s incomplete ad,juvant, repeated after a month. Allergenic specificity was tested by radioallergosorbent -.*Chervenka CH: A manual of method for the analyticat uhracentrifuge. Spinco Division of Beckman Instruments. California, 1970. tKIapper DC, Goodfriend L, Roebber M. Capra ID: Unpubtished observations. Co.

Vol. 67, No. 4, pp. 299-304

300

Goodfriend

J. ALLERGY

et al.

I 600

1

1 600

I 1000

I 1200

I 1400

I 1600

CLIN. IMMUNOL. APRIL 1981

!

VOLUME OF ELUATE (ml) FIG. 1. Molecular sieving of DEAE-fraction C through Sephadex G-50 medium. Ra3 was localized to the third chromatographic peak (CIII). Bed: 5 x 95 cm; eluting buffer: 0.05 M Tris-HCI, pH 7.6; flow: 60 mllhr.

VOLUME OF ELUATE (ml) Fffi. 2. Chromatography of fraction 2 (Clll-2). Bed: 5 x 25 cm; eluting

C-III through DEAE-Sephadex A-50. Ra3 was localized buffer: 0.001 M Tris-HCI, pH 7.6; flow: 120 mllhr.

test (RAST) inhibition using paper discs coupled with Ra3I by the method described for codfish allergen by Aas and Lundkvist.lR Duplicate aliquots (50 ~1) of phosphatebuffered saline (PBS) (pH 7.2) or varying concentrations of test inhibitor in PBS were added to the discs prior to addition of .50-j-6.1 aliquots of allergenic serum. The assay was performed according to the procedure of Phadebas RAST (Pharmacia Diagnostics AB, Sweden).

RESULTS IsoMon of ReSf and R&II

A frequently employed modification of the initial procedure’ for isolation of Ra3 was as follows. An

to peak

aliquot (25 ml) of DEAE-fraction C equivalent to 125 gm pollen was sievedthrough SephadexG-50 medium to give the Ra3 fraction CIII (Fig. 1). A pool (30 ml) of recycled Cl11 fractions corresponding to 0.5 kg pollen was equilibrated against0.001 M Tris-HCl, pH 7.6, and chromatographedthrough DEAE-Sephadex A-50 (Fig. 2). After recycling the sameion-exchanger, disc electtophoresisof the Ra3 fraction CIII-2 derived from different kilogram lots of short ragweed pollen invariably showedthe presenceof a minor component along with the anticipated Ra3 band (Fig. 3). It was found possibleto separatethe componentsby multiple cycles through SephadexG-50 superfine (Fig. 4) and

Variants

Fio. 3. Polyscrylamide fraction WI-2.

gel disc electrophoresis

allergen

Ra3

301

(pli 4.3) of 900

in this manner to obtain disc electrophoretically homogeneousRa31 and Ra311(Fig. 5). In general, yields of unresolved Ra3, Ra3I and of Ra3II were approximately 100, 30, and 10 mg/kg pollen. Sam0 prapartb

of ragweed

of Ra31 and Ra311

The molecular weights of the major and minor Ra3 compo~nts were essentially identical. Partial specific volumes of 0.734 and 0.722, corrected for 8% carbohydrate,* were calculated from the corresponding m&r amino acid compositions. The respectivevalues for dlogc/dr* obtained at equilibrium in the analytical ultracentrifuge were 0.251 and 0.260 (Fig. 6). Sub-. -*Determined” for Ra3I and assumed for Ra311. The assumption is hosed on ( 1) carbohydrate analysis of Ra3I and Ra3II isolated by preparative disc eIectrophoresi6’ showing essentially equal (though lower) carbohydrate content. and (2) concordance of the u~traceatrifigal molecular weight values with values obtained tiom the complete amino acid sequences of Ra31 and Ra311.

1000

1100

1200

VOLUME OF ELUATE (ml) FIG. 4. Separation of Ra31 and Ra3ll by gel filtration through SephadexG-50superfine. A-C, Consecutive cycles in the purification of Ra3ll (fraction Clll-2b). D, Final cycling of Ra3l (fraction Clll-2a). Bed: 5 x 95 cm; eluting buffer: 0.025 M Tris-HCI, pli 7.6; flow: 20 mlihr.

stitution into the standardequation”* gave a weightaverage molecular weight of 12,XlO d&ons for both proteins, or approximately 11,000daltorrsfor the protein moieties. The amino acid compositions of Ra31 and Ra3II (Table I) were similar, showing siogle residue differences for a number of amino acids. As noted in the table. some of these residue differences have been confirmed in studies on the compkte amino acid sequencesof Ra31and Ra3II isolated subsequentto *Chervenka CH: A manual of method for the analytical ultracentrifuge. Spinco Division of Beckman instruments. California. 1970.

302

Goodfriend

et

al.

J. ALLERGY

CLIN. IMMUNOL. APRIL 1981

-1.6--

0.0

-0.5

i

FIG. 5. Polyacrylamide gel disc electrophoresis. plus Ra3ll; B, Ra3l; C, Ra3ll.

A, Ra3l

-1.0

I

this study from a different batch of pollen.* From the sequence-derived compositions, the molecular weights of Ra31 and Ra311were calculated to be 11,306 and 11,383, respectively, in close agreement with the ultracentrifugal results. The amino acid compositional differences between Ra31and Ra311were not reflected in the antigenic or allergenic specificities of the two forms. Thus, reactions of complete identity were obtained by immunodiffusion with rabbit antiserum of Ra31or to WSR (Fig. 7). In addition, soluble Ra311was as effective as soluble Ra31in inhibiting binding to solid-phaseRa31 of IgE antibodies in three ragweed-sensitive sera (Table II).

-1,5~, 46

47

49

49

50

r2 (cm=)

FtG. 6. Least-squares plot and slope (upper graph) and Ra3ll (lower).

of logcll3

for Ra3l

(Table I) show single residuedifferencesfor a number of amino acids. Some of these have been confirmed and additional differences revealed by amino acid sequenceanalysis* (Table I). Amino acid variation has already been observed’* for short ragweed pollen allergen RaP (molecular weight 5,000 daltons). However, the variation was limited to a single residue, that is, some 30% of Ra5 molecules had DISCUSSION leucine (minor variant) instead of valine (major varThe two variants of Ra3 were purified to homogeiant) at the second N-terminal position along the polypeptide chain. Becauseof the chemical similarity neity as assessedby disc electrophoresisand linearity of these amino acids, it was obviously not possible to of the dlogc/dP plots on analytical ultracentrifugaseparatethe major and minor variants of Ra5 by the tion. On the basis of an 8% carbohydrate content, their molecular weights were identically 12,300 dalusual chromatographic procedures. In the case of Ra3, however, more extensive as well as some nontons. Thus, resolution of the variants by gel filtration conservative substitutions* in particular regions of its through SephadexG-50 supertine may be ascribed to singles*l9 polypeptide chain may have imparted suffiprotein-gel interaction stemming from possible qualitative difference in the nature of the carbohydrate cient physicochemical differencesto permit chromatographic separationof the major and minor variants. moieties or differences in amino acid composition (cf. the separation of antigens E and K”). As already noted, purified allergen Ra5 has been The amino acid compositions of Ra31 and Ra311 found to consist of molecular variants in the propor*Klapper DG, Goodfriend L, Roebber M, Capra JD: Unpublished observations.

*Klapper DG, Goodfriend L, Roebber M, Capra JD: Unpublished observations.

VOI UME NUMBER

67 4

Variants

LE I. Amino acid composition

-

Akrmber

-_

303

Found 7.2 (9) 2.7 (1)

(2) (6) (7) (7) (4)

4.2 8.6 7.0 4.9 9.9 8.2

10.5 (10) 7.1 (7) 10.4 (11) 7.0 2.0 6.6 2.7 7.7 2.8 6.9

Ra3

gm

6.4 (7)* 2.5 4.0 8.7 7.0 4.7

allergen

of Ra31 and Re341

of rsaklmrtll~

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine/2 Valine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan (Total)

of ragweed

(6) (8) (5) (4)

(10) (7)

9.1 (10)

(7) (2) (7) (3) (8) (4) (6)

6.4 2.8 6.3 3.5 7.6 2.9 6.2 ND

2.1 (3) (101)

(7) (3) (7) (3) (8) (4) (6) (3)

(101)

*\&es in parentheses arethe numberof aminoacidresiduesestablished by complete amino acid sequence determination of RF?1 and Ra3II isolated from short ragweed pollen subsequent tc: this study.

E IL Allergen specificity of Ra31 and RaFN by pet-cant RAST inhibition -. Test inhib%or cone. (pglml) 10-J Serum I

-..

lo-’

10.’

FIG. 7. lmmunodiffusion

Rasl

II

I

II

I

II

8 53 47

13 53 50

57 74 80

48 75 81

76 89 91

67 90 90

tion of 3: 1. Interestingly,

the yields of Ra3I and Ra3II were generally in the sameratio. If, as is likely, these yields reflect the actual content of the variants in the pollen, this would suggest that the major and minor variants of Ra3 and Ra5 belong respectively to maJorand minor variants of short ragweed pollen. It seemsplausible that various mutational forms of short ragweed pollen (and other pollens) have developed with predominant forms in different geographical regions, producing protein allergens of common though partially distinct primary structures and antigenic or allergenic specificity determinants. Although in this study no differential antigenic or allergenic specificities were observedfor Ra3I and Ra311,the assays were clearly limited in scope and more extensive

analysis of antigen Ra31 and Ra311 using rabbit antiserum to Ra31 (antigen cont., 0.2 mglml) and to WSR (antigen cont., 0.1 mgiml).

comparison seemswarranted. Of interest in this connection is the finding of yet a third major variant of Ra3 (“NIH-Ra3”) having a third pattern of amino acid sequence in the same (restricted) regions of the polypeptide chain.lg Although the NIH and our prep arations of Ra3 gave reactionsof complete identity on immunodiffusion with Ra3-specificantiserum,* some evidence appearsto have been obtained by leukocyte histamine releaseassayfor markeddifferences in their allergenic specificities.t -*Roebber M, Goodfriend L: Unpublished observations tLichtenstein LM: Personal communication. REFEflEMCES I. Underdown L3.J.Goodfriend L,: Isolation and characterization of an allergen from shon ragweed pollen. Biochemistry 8:980. 1%9. 2. Adolphson C. G&friend L, Gleich GJ: Reacttvity of ragweed

304

3.

4.

5.

6.

7.

8.

9.

Goodfriend

et al.

allergens with IgE antibodies. J ALLERGYCLIN IMMUNOL 62:197, 1978. Marsh DG, Bias WB, Hsu SH, Goodfriend L: Associations between major histocompatibility (HL-A) antigens and specific reaginic antibody responses in allergic man, in Goodfriend L, Sehon AH, Orange RP, editors: Mechanisms of allergy: Reagin-mediated hypersensitivity, New York, 1973, Marcel Dekker, Inc., pp. 113-129. Choudhury AM, Goodfriend L: Differentiation between carrier and allergenic determinants in ragweed allergens Ra3 and Ra5. Fed Proc 37:1553, 1978. (Abst.) Marsh DG, Goodfriend L, Bias WB: Basal serum IgE levels and HLA antigen frequencies in allergic subjects. I. Studies with ragweed allergen Ra3. Immunogenetics 5:217, 1977. Marsh DG, Chase GA, Freidhoff LR, Meyers DA, Bias WB: Association of HLA antigens and total serum immunoglobulin E level with allergic response and failure to respond to ragweed allergen Ra3. Proc Nat1 Acad Sci USA 76:2903, 1979. Bias WB, Hsu SH, Meyers DA, Goodfriend L, Marsh DG: HLA associations with chemically defined ragweed pollen components. Transplant Proc 11:1853, 1979. Roebber M: Isolation, structure and activity of ragweed pollen antigens Ra3, Ra4 and Ra5. Doctoral thesis, McGill University, Montreal, 1975. Reisfeld RA, Lewis UJ, Williams DE: Disc electrophoresis of basic proteins and peptides on polyacrylamide gels. Nature 195:281, 1962.

J. ALLERGY

CLIN. IMMUNOL. APRIL 1981

10. Koening R, Stegemann H, Francksen H, Paul HL: Protein sub-units in the potato virus x-group: Determination of the molecular weights by polyacrylamide electrophoresis. Biochem Biophys Acta 207: 184, 1970. 11. Lapkoff CB, Goodfriend L: Isolation of a low molecular weight ragweed pollen allergen: Ra5. Int Arch Allergy Appl. Immunol 46:215, 1974. 12. Cohn EJ, Edsall JT: In Cohn EJ, Edsall JT: Proteins, amino acids and peptides as ions and dipolar ions. New York, 1943, Reinhold Publishing Corp. 13. Gibbons RA: In Gottschalk A, editor: Glycoproteins. New York, 1966, Elsevier Publishing Co. 14. Kabat EA, Mayer MM: Experimental Immunochemistry. Springfield, Ill., 1961, Charles C Thomas, Publisher. 15. Beaven GH, Holiday ER: In Anson ML, Bailey K, E&all JT, editors: Advances in protein chemistry. New York, 1952, Academic Press, Inc., vol. 7. 16. Aas K, Lundkvist U: The radioallergosorbent test with a purified allergen from codfish. Clin Allergy 3:225, 1973. 17. King TP, Norman PS, Lichtenstein LM: Isolation and characterization of allergens from ragweed pollen. IV. Biochemistry 6:1992, 1967. 18. Mole LE, Goodfriend L, Lapkoff DB, Kehoe JM, Capra JD: The amino acid sequence of ragweed pollen allergen Ra5. Biochemistry 14:1216, 1975. 19. Klapper DG, Goodfriend L, Capra JD: The amino acid sequence of ragweed allergen Ra3. Biochemistry 19:5729,1980.