<arttitle> Group-2/3 Allergens of Grass Pollen Have Cell
Wall-Loosening Activity Characteristic of -Expansins.
<aug> Lian-Chao Li, Mark W. Shieh & Daniel J. Cosgrove
<aff> Department of Biology, 208 Mueller Laboratory, Pennsylvania State University,
University Park, PA 16802, USA
<abs> Group-2 and group-3 allergens from grass pollen are major elicitors of hay fever
and seasonal asthma in humans1,2, but the native activity and biological roles of these
proteins have not been examined. These small (~11-kD) proteins1,3-5 have sequence
similarity to the C-terminus of expansins6. Expansins induce extension and stress
relaxation of plant cell walls in a unique manner7-10 and function in a variety of plant
developmental processes where cell wall loosening is important11-18. Here we show that
both native and recombinant group-2/3 allergens have expansin-like cell wall loosening
activity. These small proteins appear to be a unique evolutionary innovation of grasses,
with a specialized role in pollen function. Our results indicate that the characteristic wallloosening action of expansin is attributable to its C-terminal domain.
<p> Although group-2 and group-3 grass pollen allergens are distinguished by pI and
immuno-cross reactivity19, accumulating sequence information indicates they belong to the
same protein family, which we refer to as group-2/3 allergens. Genes for group-2/3 allergens
encode a protein with a signal peptide and a mature protein having up to 42% identity with
domain 2 of expansins, with the greatest similarity to group-1 allergen sub-class of expansins (Fig. 1). Expansins loosen plant cell walls without hydrolysis of the major structural
polymers of the wall, apparently by disrupting adhesion of key wall polysaccharides to one
another20,21. Two expansin families (- and -) are currently recognized, each containing an
N-terminal domain, homologous with the catalytic domain of glycosyl hydrolase family 45
(GH45) enzymes, and a C-terminal domain, hypothesized to be a polysaccharide-binding
1
domain 8. Although the GH45-like domain was previously suspected to be expansin’s
catalytic domain, our new findings change our thinking about the domain responsible for the
characteristic action of expansin and gives important clues to the biological role of the group2/3 allergens.
We purified proteins from ryegrass (Lolium perenne L.) pollen extracts using three
sequential chromatographic steps combined with assays of wall extension activity 8. A single
peak of activity was eluted from an SP-Sepharose column (Fig. 2a) and sub-fractionation on a
CM-silica based HPLC column yielded multiple active fractions (Fig. 2b), which were further
separated by gel filtration chromatography to yield pure Lol p 3 and Lol p 1 (Fig. 2c-d). These
group-3 and group-122,23 allergens from ryegrass4,19 were identified by SDS-PAGE and
immunoblot analysis. Lol p 3 was definitively identified by its N-terminal sequence
(TKVDLTVEKGSDAKTLVLNI) and by its molecular mass (10,908.1 dalton versus the
predicted value of 10,908.8 dalton). Lol p 1 is a member of the -expansin family{Cosgrove,
Bedinger, et al. 1997 3296 /id}{Li & Cosgrove 2001 4250 /id} and thus was expected to have
wall extension activity, but this was not expected for Lol p 3.
To verify the wall-loosening activity of Lol p 3, we cloned its DNA into the pET22b(+)
expression vector and confirmed inducible wall-extension activity in bacterial extracts (Fig.
3a). Additionally, we found similar wall-extension activity for native Phl p 2, a group-2
allergen purified from timothy grass (Phleum pratense L.) pollen, and for Zea m 3, a group-3
allergen isolated from maize (Zea mays L.) pollen. Thus we conclude that group-2/3 allergens
indeed possess expansin-like cell wall loosening activity. Further extensometer assays showed
that Lol p 3 induced extension of cell walls from many grass species, whereas it was
ineffective with a variety of dicot cell walls (Fig. 3b).
2
A second hallmark of expansin action is enhancement of wall stress relaxation, and Lol
p 3 substantially increased stress relaxation of grass walls (Fig. 3c). For this assay, which
measures the mobility of load-bearing polymers in the cell wall, the specimen is rapidly
extended, then held at constant length and the subsequent decay in holding force is measured.
In contrast to its action on grass walls, Lol p 3 had negligible effect on stress relaxation of
dicot walls (e.g., cucumber hypocotyl walls). This selectivity for grass walls, in both extension
and stress relaxation assays, is similar to that found for the group-1 pollen allergen class of expansins21,24. Grass walls are notable for their distinctive wall composition25, with reduced
amounts of xyloglucans and pectins and increased amounts of arabinoxylans. Thus, group-2/3
allergens, like -expansins, appear to act on a wall polymer with particular structural
significance to grass cell walls.
Wall extension activity of Lol p 3 was saturable and was most active at pH 5.5 (Fig. 3de), which is close to the wall pH of many plant tissues26. The sharp reduction in activity at pH
< 4.5 is very different from that found for -expansins, which have higher activity at pH 3-4
than at pH 5-69,27. This property is germane to the “acid growth” process in plants26, wherein
plant cell enlargement and wall loosening are stimulated by low cell wall pH. Because of its
pH dependence, Lol p 3 is not a good candidate as an acid-growth agent. Instead, its pH
dependence is centered on the normal pH of the cell wall.
Lol p 3 was also found to have strong synergistic (more than additive) activity with the
-expansin Lol p 1 (Fig. 3f). This result may indicate that the two proteins act on different cell
wall components which combinatorially determine the mechanical properties of the grass cell
wall. Alternatively, the proteins may interact so as to stimulate their wall loosening activities.
In further studies we found that Lol p 3 had the following unusual properties that are
also characteristic of expansins: First, Lol p 3 weakened pure cellulose paper, whose
3
mechanical strength derives from noncovalent bonding between cellulose fibres. This result
indicates that Lol p 3, like expansin10, can disrupt the noncovalent bonding between glucans.
Second, Lol p 3 apparently binds tightly to cell walls, because walls pretreated with Lol p 3
continued to extend after nonbound Lol p 3 was removed and replaced with buffer alone.
This behaviour is similar to that of - and -expansins, which are not readily removed from
plant cell walls after binding20,21. Third, the extension ability of walls pre-treated with Lol p 3
was eliminated by subsequent treatment with protease. Because wall structural proteins are
not important determinants of cell wall extensibility21, we interpret the effect of protease
digestion to be due to inactivation of wall-bound Lol p 3. We conclude therefore that Lol p 3
did not irreversibly weaken the cell walls and that continued Lol p 3 action was required for
continued wall extension. This also is similar to expansin action.
In searches of the public sequence databases, we identified 12 group-2/3 allergens. All
were from grasses, and analysis of the maize and rice EST databases indicates that they are
predominantly, if not exclusively, expressed in pollen, not in vegetative tissues. It is
noteworthy that the Arabidopsis genome does not contain any genes encoding group-2/3
allergens, nor are any found in current EST collections from dicots. Thus, their phylogenetic
distribution indicates that group-2/3 allergens evolved only in the grass lineage, most likely by
deletion of domain 1 from a -expansin gene (Fig. 1).
Our experimental results imply that the characteristic action of expansin on cell wall
extensibility and stress relaxation is principally due to the action of domain 2. However,
except for the single case of the grass group-2/3 pollen allergens, the GH45-like domain 1 of
expansin has apparently been preserved throughout plant evolution (~500 million years). This
in turn implies that the GH45-like domain has an important undiscovered role in expansin
function, perhaps to amplify, regulate, or localize the cell-wall loosening action of domain 2.
4
<meth1ttl> Methods
<meth1hd> Proteins were extracted and purified from grass pollen and analyzed by
SDS/PAGE and immunoblotting as described21. Purified Lol p 3 and Zea m 3 were analyzed
by ESI-TOF mass spectrometry and sequenced by automated Edman degradation.
Heat-inactivated wall specimens were prepared from coleoptiles and hypocotyls of etiolated
seedlings and from maize silks as described21. For extension assays, specimens were clamped
in an extensometer with 20-g load (5 g for silks) in 50 mM sodium acetate, pH 4.5. Stress
relaxation assays were performed as described28, except that walls were pretreated for 10 min
in 50 mM sodium acetate, pH 4.5, with or without 50 g/mL Lol p 3, and then stored on ice
briefly before the assay.
Cloning and recombinant expression of Lol p 3. The protein sequence for Lol p 3 was
known4, but not its nucleotide sequence. Therefore we used PCR to amplify the sequence
encoding the mature protein, using a degenerate 24-base sense primer [5’-ACN(ACGT)A
AR(AG)G TN(ACGT)G AY(CT)Y(CT) TN(ACGT)A CN(ACGT)G TN(ACGT)G AR(AG)3’], a 24-base antisense primer [5’-CY(CT)Y(CT)A R(AG)TT R(AG)TA Y(CT)TC
N(ACGT)GG N(ACGT)GT R(AG)TA N(ACGT)GT-3’] and genomic DNA as template. The
294-bp PCR product was cloned into pCR2.1-TOPO and sequenced. The insert was digested
with Nde 1 and Eco R1, ligated into pET22b(+) (Novagen, Inc., Madison, WI, USA ) and
expressed in E. coli Novagen AD494 (DE3) competent cells, following the manufacturer’s
protocol for induction with 1 mM isopropyl -D-thiogalactoside. Cells were grown for an
additional 3 h, centrifuged, resuspended in 10 mL of cold 1% CHAPS with 5 mM
dithiothreitol and 1/5 tablet of the complete protease inhibitor cocktail (Roche) and lysed by
ultrasonication. The lysate was brought to 10 mM sodium acetate, pH 4.5, sedimented at
12,000g for 20 min, chromatographed on a CM-Sepharose column and the active fraction was
desalted by ultrafiltration.
5
<received> Received ________2003 and accepted ___________2003.
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7
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Acknowledgements
<ack> This work was supported by grants from the U. S. Department of Energy and by the U. S. National
Institutes of Health. We thank Dr. A. Daniel Jones III of the PSU Mass Spectrometry Center for ESI-TOF mass
spectra analysis and Dr. Anne Stanley of the PSU Macromolecular Core Facility for N-terminal sequence
analysis of Lol p 3 and Zea m 3. Daniel M. Durachko provided expect technical assistance. Dr. David G. Klapper
8
(University of North Carolina School of Medicine, Chapel Hill, NC) generously provided mouse monoclonal
antibody (anti-site D) raised against Lol p 129. Rabbit polyclonal antibodies, raised against natural Lol p 2 or
recombinant Phl p 2, were kindly supplied Dr. Alessandro Sidoli (Department of Biological and Technological
Research, San Raffaele Scientific Institute, Milano Italy) and Dr. Rudolf Valenta (Institute of General and
Experimental Pathology, AKH, University of Vienna, Vienna, Austria).
Competing interests statement
The authors declare that they have no competing financial interests.
<corr> Correspondence and requests for materials should be addressed to D.J.C. (e-mail: dcosgrove@psu.edu).
The sequence for Lol p 3 is deposited at GenBank with the accession number AY135645.
9
<LEGEND> Figure 1 Structural comparison of group 2/3 allergens and expansins.
Group 2/3 allergens are homologous with domain 2 of - and -expansins, with
greatest similarity to the group-1 allergen subclass of -expansin. SP=signal
peptide; PDB=polysaccharide binding domain.
Figure 2 Purification of group-2/3 and group-1 allergens from ryegrass pollen. a,
Fractionation of ryegrass pollen extract by SP-Sepharose Fast Flow chromatography
in 20 mM sodium acetate, pH 4.5, eluted with a 0~500 mM NaCl linear gradient from
120-240 min. b, Active fractions from a were fractionated on a silica-based CMHPLC column with elution at 1 mL/min with a 0~650 mM NaCl, 20 mM sodium
acetate, pH 4.5 linear gradient in 50 min. c, Lol p 3 purification from b by highperformance gel filtration chromatography. Inset shows Coomassie-stained
SDS/PAGE (S) and western blot (W) probed with antibody against group 2 allergens.
d, Lol p 1 purification by high-performance gel filtration chromatography. Inset
shows Coomassie-stained SDS/PAGE (S) and western blot (W) probed with
antibody against Lol p 1.
Figure 3 Lol p 3 action on plant cell walls. a, Recombinant Lol p 3 has wall
extension activity. At the arrow, partially purified protein (250 g/mL) from E. coli with
or without the Lol p 3 expression vector. b, Lol p 3 (50 g/mL) promotes extension
of cell walls from grasses but not dicots. Grass walls included maize silks and wheat
coleoptiles (shown), with similar results from coleoptiles of barley (Hordeum sativum
L.), rice (Oryza sativa L.), and oat (Avena sativa L.). Dicot walls include hypocotyls of
cucumber (Cucumis sativus L., shown ) as well as soybean (Glycine max L.), tomato
(Lycopersicon esculentum Mill.), rape (Brassica napus L.), pea (Pisum sativum L.)
and pepper (Capsicum annuum L.). c, Native Lol p 3 (50 g/mL) enhances stress
relaxation of wheat coleoptile walls. Each curve is the average of 10 independent
measurements. d, Wall-extension activity of Lol p 3 as a function of protein
10
concentration, assayed with wheat coleoptile cell walls in 50 mM 3,3-dimethylglutaric
acid, pH 5.5. Mean +/- standard error of five replicates e,
pH dependence of wall-
extension activity of Lol p 3 (50 g/mL), assayed with wheat coleoptiles in 50 mM
3,3-dimethylglutaric acid at different pH values. Mean +/- standard error of five
assays.
f, Synergism between Lol p 3 and Lol p1 in wall extension assays. Lol p 1
(20 g/mL) and Lol p 3 (10 g/mL) were added singly or in combination to wheat
coleoptile cell walls in 50 mM sodium acetate, pH 4.5. Means +/- standard errors of
five replicates.
11