Isoforms of Bet v 1, the Major Birch Pollen Allergen, Analyzed by Liquid Chromatography, Mass Spectrometry, and cDNA Cloning

doi:10.1074/jbc.270.6.2607

Isoforms of Bet v 1, the Major Birch Pollen Allergen, Analyzed by Liquid Chromatography, Mass Spectrometry, and cDNA Cloning (*)

From the (1)Institut für Allgemeine und Experimentelle Pathologie, Universität Wien, Währinger Gürtel 18-20, A-1090 Vienna, Austria, the
(2)Institut für Genetik und Allgemeine Biologie, Universität Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria, the
(3)Institut für Mikrobiologie und Genetik, Universität Wien, Dr. Bohrgasse 7, A-1030 Vienna, Austria, the
(4)Institut für Analytische Chemie, Universität Wien, Währingerstraβe 38, A-1090 Vienna, Austria, and the
(5)Ernst Böhringer Institut für Arzneimittelforschung, A-1120, Vienna, Austria

↵^¶ Present address: Inst. f. Genetik u. Allg. Biologie, Universität Salzburg, Hellbrunnerstr. 34, A-5020 Salzburg, Austria.

** To whom correspondence and reprint requests should be addressed.

Abstract

Bet v 1, the major allergen of birch pollen, displays a considerable degree of heterogeneity. Several charge variants have been detected by two-dimensional IgE immunoblots and isoelectric focusing techniques. This heterogeneity has been attributed to glycosylation (or other post-translational modifications) or to isogenes coding for Bet v 1 isoforms and/or allelic variants. However, until now, only limited structural data for Bet v 1 have been published. Recently, we described the expression, purification, and immunological properties of recombinant Bet v 1 (rBet v 1) produced in Escherichia coli as a non-fusion protein (Ferreira, F. D., Hoffmann-Sommergruber, K., Breiteneder, H., Pettenburger, K., Ebner, C., Sommergruber, W., Steiner, R., Bohle, B., Sperr, W. R., Valent, P., Kungl, A. J., Breitenbach, M., Kraft, D., and Scheiner, O.(1993) J. Biol. Chem. 268, 19574-19580). Here, we present a more detailed structural characterization of Bet v 1 by both cDNA cloning and mass spectrometry. Thirteen different cDNA clones coding for Bet v 1 isoforms were obtained by polymerase chain reaction amplification of birch pollen cDNA with a sequence-specific 5′-terminal primer and a nonspecific 3′-terminal primer or by immunological screening of a birch pollen cDNA library. These isoforms are referred to as Bet v 1b to Bet v 1n, whereas the previously isolated Bet v 1 cDNA (Breiteneder, H., Pettenburger, K., Bito, A., Valenta, R., Kraft, D., Rumpold, H., Scheiner, O., and Breitenbach, M.(1989) EMBO J. 8, 1935-1938) is now referred to as Bet v 1a. High performance liquid chromatography and plasma desorption mass spectrometry of proteolytic fragments of purified natural Bet v 1 (nBet v 1) and rBet v 1a were used to (i) confirm the primary structure of all Bet v 1 isoforms and (ii) to investigate any possible postsynthetic modifications on rBet v 1a or on the natural mixture of isoallergens obtained from birch pollen. Except for the cleavage of initiating methionine, no postsynthetic modifications were found in either nBet v 1 or rBet v 1a.

Footnotes

↵^§ The first two authors contributed equally to this paper.
↵* This work was supported by Grants P10019-MOB (to F. F. and M. B.) and S6002-BIO (to O. S.) from the Austrian Fonds zur Förderung der Wissenschaftlichen Forschung (F.W.F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) 77200, 77265-77270[GenBank], 77271-77273[GenBank], 77274, 81972, and 82028.
↵¹ The abbreviations used are:

Bet v

Betula verrucosa

Aln g

Alnus glutinosa

Car b

Carpinus betulus

Cor a

Corylus avellana

HPLC

high performance liquid chromatography

nBet v 1

natural Bet v 1

PCR

polymerase chain reaction

PDMS

plasma desorption mass spectrometry

rBet v 1

recombinant Bet v 1.
- Received August 15, 1994.
- Revision received October 18, 1994.