Annexin Characterization

Isolation, Identification, and Analysis of a cDNA encoding an Annexin-like Protein from Arabidopsis thaliana




Introduction

Calcium ions play a vital role in the complex cellular signaling pathways within plant cells1. Thus, the regulation of free calcium within cells is of paramount importance in the control of cellular signaling. A major mechanism of such regulation is through calcium-binding proteins that sequester the amount of free calcium available for signaling activation2.

Annexins belong to a family of calcium-binding proteins found associated with the plasma membrane of cells. Annexins were first characterized in animal cells where their specific function is still unclear. However, research has revealed that annexin proteins bind specifically to phospholipids, particularly the phospholipid bilayer of the cell plasma membrane3. Interestingly, annexin proteins must first bind to acidic phospholipids before calcium binding at physiological levels can take place3. It has been speculated that annexin proteins may play a variety of critical roles in plant cells, from regulation of calcium-dependent biochemical signaling processes to phospholipid metabolism4. Research into the specific roles of annexin proteins in plant cells is an explosive area of research in current plant biology4.

A large number of plant annexins have been isolated and studied, including Zea mays (corn)14, Capsicum annuum (pepper)15, Gossypium hirsutum (cotton)16, and Arabidopsis thaliana (thale cress)13. The annexin proteins from these and other plants all show similar patterns in amino acid sequence. In particular, all known annexin proteins show 70 amino acid repeat domains, within which there are smaller, highly conserved consensus regions3.

In this paper, we report the isolation, identification, and analysis of a novel annexin-like protein from A. thaliana. We sequenced a cDNA clone from A. thaliana and through homology search identified it encoding an annexin-like protein. The amino acid sequence seemed to be moderately conserved (60-69%) between other plant annexins. However, alignment with human annexin showed considerably lower homology (24%). Southern hybridization data pointed to the annexin gene family being relatively small in A. thaliana. In addition, Northern hybridization experiments revealed tissue-specific expression of annexin in flowers, and an increase in annexin mRNA transcription after wounding. The deduced amino acid sequence was also used for protein motif and pattern searches. The annexin protein was found to have four phosphorylation sites, and an overall hydrophilic, non-antigenic structure comprised primarily of alpha-helices.





Materials and Methods


Isolation, restriction digestion, and ligation of cDNA insert:

The pZL1 (Life Technologies) plasmid containing the cDNA insert of interest was isolated from E. coli using an alkaline-lysis method5. The plasmid was then digested with the restriction enzymes EcoR1, BamH1, and HindIII (Promega), in order to obtain the cDNA insert for ligation6. The pBluescript (Stratagene) plasmid vector was also digested by these same restriction enzymes. The cDNA clone was excised from a preparatory agarose gel and purified. A standard protocol was then used to ligate the cDNA clone into the pBluescript vector6. The plasmid construct insert was isolated and purified from transformed E. coli for sequencing8.


Sequencing and computer analysis:

The cDNA insert in the vector pBluescript was denatured into single stranded DNA in preparation for sequencing9. The DNA was then sequenced using the dideoxy chain termination technique using T7 and T3 primers10. The sequence obtained from manual sequencing was analyzed using MacVector and SeqApp computer software11. Open reading frame (ORF) analysis, translation, and restriction mapping was done using MacVector, while homology alignment was carried out using SeqApp software. Database analysis and motif searches of the DNA and amino acid sequences was done using BLAST, FASTA, Motif explorer, and other programs at the National Center for Biotechnology Information (NCBI), Baylor College of Medicine (BCM), and Arabinet internet sites12.


Genomic DNA isolation and Southern hybridization:

Genomic DNA from A. thaliana was isolated by the standard N2(l)/SDS/phenol technique17. The DNA was then digested using the restriction enzymes EcoR1, HindIII, and BamHI and fractionated on a 0.8% agarose gel. The DNA bands were then transferred to a Zetaprobe nylon membrane (BioRad) using capillary transfer18.

A complete annexin cDNA probe was labeled using alpha-[P32] dATP by a random primer method (Ambion). The probe was then allowed to hybridize to the genomic DNA in a 0.25M Na2HPO4, 1mM EDTA, and 7% SDS solution overnight. A low stringency wash with 40mM Na2PO4, 1mM EDTA, and 5% SDS was performed at 60oC17. The blot was exposed until the radioactive bands of interest were visable.


Isolation of total RNA and Northern hybridization:

Total RNA from the leaf, flower, stem, and whole plant of A. thaliana was isolated using N2(l)/SDS, and phenol/chloroform17. This same procedure was repeated to isolate RNA from plants 0,1,5, and 8 hours after wounding. The RNA was size fractionated on a formaldehyde-agarose gel and then transferred to a Zetaprobe nylon membrane using capillary transfer. The RNA was hybridized against the same annexin cDNA probe, and the Southern hybridization procedure above was repeated.




Results


Isolation, restriction digestion, and ligation:

We were able to cleanly isolate the pZL1 plasmid containing the cDNA insert of interest. Restriction digestion yielded the cDNA clone, and the restriction enzymes EcoR1 and BamHI were determined to optimally cut the cDNA insert out of the pZL1 plasmid vector. The data obtained also showed that the cDNA insert itself did not contain any EcoR1 or BamH1 restriction sites, and we were able to estimate the size of the insert at approximately 1.2 kb.


Sequencing and analysis:

We sequenced both strands the cDNA insert using both T7 and T3 primers. The main ORF was found using MacVector program from base 3 to base 809, and consisting of 269 amino acids (See Fig. 1). The DNA sequence of the insert was also used to determine the restriction map of the insert (See Fig. 2). The sequence of the cDNA insert was then analyzed using a BLAST search at NCBI, and was identified as an A. thaliana annexin-like gene (score=546, p=3.7e-35). This identification was confirmed through a BLAST search at NCBI of the deduced amino acid sequence. Again, the amino acid sequence was identified as coding for an annexin-like protein (score=875, p=4.2e-115). Data for the amino acid sequence of other similar plants and human annexin proteins was used for a homology analysis using the SeqApp program. The homology between A. thaliana and G. hirsutum (69%), C. annuum (62%), Z. mays (60%), and human (24%) annexins are shown in Fig. 3. Motif searches at the BCM and Arabinet internet sites revealed a number of phosphorylation sites (See Fig. 8), and other unique motifs. Protein secondary structure predictions at the BCM internet site and with MacVector program found the annexin-like protein to be largely alpha-helical (See Fig. 6). MacVector also predicted the protein to be hydrophilic, and have a low antigenicity (See Fig. 7).


Southern and Northern hybridizations:

The Southern hybridization results are shown in Fig. 4. The fragmentation pattern revealed four bands, with two for EcoR1, and one each for BamHI and HindIII. EcoR1 produced bands of 0.92 kb and 1.6 kb, while BamHI and HindIII produced bands of 2 kb and 3.7 kb respectively.

Fig. 5 shows the results obtained from the Northern hybridization experiment with stem, flower, leaf, and whole plant tissues from A. thaliana. Also, the results of the wounding experiment with plant samples 0,1,5, and 8 hours post-wounding are shown. There is a single band of approximately 1.30 kb from the plant flower. The size of the mRNA hybridized agrees nicely with the annexin cDNA, verifying that the entire annexin gene was probed. The wounding experiment revealed that levels of annexin mRNA, again with an approximate size of 1.34 kb, increased to a maximum at 5 hours post-wounding, and then decreased slightly over the next 3 hours.




Discussion

Arabidopsis thaliana has a relatively small genome and is a primary model organism in plant genetics13. Thus, a fundamental understanding of its internal biochemistry is important in such research. Our results conclusively identified the cDNA insert we obtained as coding for a novel annexin-like protein. These results have interesting implications in terms of the control of plant physiology and cellular signaling. As discussed earlier, annexin proteins play an critical role in the control of calcium-dependent physiological processes3. Hence, the fact that annexin-like proteins are present in A. thaliana plant cells implies that a mechanism for such control is present within the organism. Furthermore, annexin is thought to be intimately involved in plant signaling pathways via binding to plasma membrane phospholipids3. Such activity suggests possible transmembrane signal transduction pathways which lead to modification of annexins and further downstream affects.

Some evidence for possible modification of annexin proteins was found through motif searches. The large number of phosphorylation sites indicates that reversible phosphorylation, and the balance between protein kinase and phosphatase activity, may be a major mechanism of annexin regulation. Also, these findings enhance the idea of annexin¼s role in signaling, mostly since protein modification, which usually leads to conformational changes in the protein, is highly prevalent in such pathways2. In addition, computer analysis of the annexin amino acid sequence found it to be hydrophilic, non-antigenic, and alpha-helical. Since annexin is associated with plasma membranes, and not an integral protein, the hydrophilic alpha-helices would come in contact with the cytosol. This characteristic is found in a wide array of signaling molecules, including G-proteins, and lends further evidence supporting annexin¼s role in intracellular signaling pathways2.

Based on its role in cells, annexin would seemingly be well conserved throughout nature. Although not conclusively, our homology alignment data does support this hypothesis. Annexin proteins from different plants do show homology in the range of 60-69%, which is significant enough to warrant phylogenetic analysis. However, the homology between A. thaliana and human annexin proteins is far less (24%), indicating that annexin structure may be quite species specific.

The expression of our annexin-like protein in A. thaliana seems to be specific for the flowers. Given its small gene family, it seems logical for annexin to be expressed only in specific plant tissue. Moreover, our data reveals that annexin may play a role in a plants defense and repair response to wounding. This result, coupled with the evidence of multiple phosphorylation sites, adds to the notion that annexin expression is tightly controlled within plants19. Hence, the apparent tissue specificity we observed is most likely consequence of intracellular control of gene expression20.

The novel annexin-like protein we studied could potentially play a wide variety of roles within plant cells. Besides binding calcium, annexin seems to be involved in intracellular signaling and plant defense and repair response. Future work in this area will involve the expression of the annexin gene and isolation of the annexin-like protein. Biochemical investigation of the protein product and Western hybridization should reveal more about the fundamental role of annexin in plant cells.




Acknowledgments

We would like to thank Yew Lee for his assistance during the experimentation and Dr. K. Sathasivan for his outstanding advice, assistance, and interesting conversation throughout the project. This page was created using PageSpinner for Macintosh.




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