| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 8, 2002 as doi:10.1096/fj.01-0940fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Nematology Laboratory, USDA, ARS/PSI, Beltsville, Maryland, USA; and
* Department of Neuroscience, Georgetown University Medical Center, Washington, DC, USA
3Correspondence: Nematology Laboratory, USDA, ARS/PSI, 10300 Baltimore Ave., Bldg. 011A, Beltsville, MD 20705-2350, USA. E-mail: kovaleve{at}ba.ars.usda.gov
SPECIFIC AIMS
There is a dire need to develop novel strategies for controlling the multibillion dollar crop losses caused by plant-parasitic nematodes and to decrease the use of harmful chemicals. The identification of genes involved in crucial developmental stages provides information on potential targets, making it possible to change from chemical screening to target hunting, offering better ways to design and optimize methods of biologically based nematode control. We have identified the putative propeptide convertase 2 (PC2), known as a crucial enzyme for activation of neuropeptide and neurohormone precursors, from the plant-parasitic nematode Heterodera glycines.
PRINCIPAL FINDINGS
1. Screening of a cDNA library resulted in isolation of the full-length clone encoding a PC2-like peptidase of H. glycines (HglPC2), the first convertase from any plant-parasitic nematode
Sequence analysis of the coding region demonstrated a single open reading frame of 2013 nucleotides predicted to encode a 671 amino acid residue protein with molecular mass of 74.2 kDa. The deduced protein has strong amino acid homology to all known PC2s, including human, and shares the major structural characteristics. It consists of five main parts: signal peptide with transmembrane region; prosegment; catalytic domain (S8) with D/H/S catalytic triad, PC2-specific residue, and 7B2 binding sites; P domain (with RRGDT pentapeptide); and carboxyl terminus (Fig. 1
). The strongest amino acid homology was found to PC2-like enzymes of Caenorhabditis elegans (KPC-2) and Drosophila melanogaster (amontillado). Phylogenetic relatedness of HglPC2 to known PC2s from six invertebrates and eight vertebrates is illustrated in Fig. 2
. The tree shows the early split of vertebrates and invertebrates with a separate branch for mollusks. The position of H. glycines and another nematode C. elegans is close to insects and crayfish. The amino acid homology of the catalytic domain of HglPC2 to Homo sapiens PC2 and C. elegans KPC-2 is very high (74 and 88%, respectively), whereas homology to other nematode convertases (not PC2-like) was markedly lower (48, 32, 41% for KPC-1, KPC-3, and KPC-4).
|
|
2. Comparison of primary structure of all distinct PC2s known from 15 different species revealed striking similarity in overall composition of the molecule, especially its catalytic domain, with one exception
The amino acid sequence homology between HglPC2 and those of related enzymes from various species was very high for the whole molecule (from 75% for C. elegans to 55% for five mammals, including humans) and even higher for the more conserved catalytic domain (88–74%). The length of the catalytic S8 domain varied very little among all species (<1%). The distances between potential cleavage sites for the excision of the prosegment and active catalytic sites (aspartic acid, histidine, and serine) as well as the location of PC2-specific residues (oxyanion hole, recognition and binding sites for chaperone neuropeptide 7B2) were determined very strictly, and their variations did not exceed 3%. Direct amino acid patterns for active catalytic sites were almost identical for all species. This highly conserved primary structure suggests important tertiary requirements for stability and functional activity of the enzyme. In contrast, the catalytic domain of HglPC2 contained a unique additional region (position 438–466) in which the pattern typical for the S8 domain is broken (Fig. 1)
. This ‘insert’ divides the catalytic domain of HglPC2 into two subunits, and its amino acid sequence is not similar to any known fingerprints or proteins. The PC2 of C. elegans (and the nearly identical hypothetical protein C51E3.7B) has an insert with similar length and placement to HglPC2, but a different sequence. Analogous inserts were also detected in PC2-like fragments (EST database) from three different animal parasitic nematodes. Significantly, the catalytic domains of nematode sequences for proconvertases other than PC2 type did not contain such inserts.
3. Nucleotide sequence analysis depicted a (G+C) richness of HglPC2 with more extreme usage of (G+C) in the synonymous codon position, whereas the PC2 of C. elegans was oppositely biased
The (G+C) content for the coding region of HglPC2 was GC = 0.556 and GC3s = 0.615 (0.473 and 0.384 for C. elegans). The relative amount of purines and pyrimidines in the silent position of HglPC2 was about equal, as were the ratios between A and T or C and G. The amount of purines in PC2 of C. elegans was relatively less than the amount of pyrimidines, and C was used almost twice more often than G in the synonymous position. Amino acid usage had a higher level of (Pro+Leu+Arg) and a lower level of (Ile+Asp+Asn+Phe) than in the PC2 of C. elegans. Codon usage was especially different in several amino acids, such as Pro, Arg, Gly and Leu. The higher RSCU values were observed for G/C-ending codons in HglPC2 and for A/T-ending codons in PC2 of C. elegans. Cluster analysis of EST fragments for PC2-like coding regions from other nematodes, followed by nucleotide analysis, was done for two more available plant nematodes: Globodera rostochiensis and Meloidogyne incognita. A comparison of corresponding fragments depicted a similar base composition for G. rostochiensis (GC=0.552, GC3s=0.637 for a 210 amino acid region covering most of the S8 domain, for which HglPC2 values were 0.537 and 0.596 respectively), but not for M. incognita (GC=0.402, GC3s=0.191 for a 172 amino acid region covering the P domain and part of carboxyl terminus, for which HglPC2 values were 0.576 and 0.650). Amino acid homology of corresponding regions in both cases was very high (84 and 78% for G. rostochiensis and M. incognita).
4. Expression of HglPC2 has a temporally restricted pattern
Two pairs of primers designed to amplify HglPC2 from a cDNA library provided positive PCR products from cDNA samples of J2 juveniles and eggs of H. glycines. Similar products were detected from cDNA of mixed stages of C. elegans. The same primers did not yield any products from cDNA of adult stages (young or mature females) of H. glycines. As a control of female cDNA quality, we did detect cDNA for two abundant proteins, actin and heat shock protein 70, in amplifications simultaneously performed with HglPC2 amplifications.
CONCLUSIONS AND SIGNIFICANCE
The new member of the PC2 family isolated from soybean cyst nematode belongs to the class of evolutionarily conserved Kex2/subtilisin-like proprotein convertases, with similar structure from nematodes to mammals. PC2 (E.C.3.4.21.94), expressed selectively in neural and endocrine tissues, is known as a key enzyme for proteolytic maturation of proteins in the regulated secretory pathway. The involvement of PC2 in distinct metabolic pathways is different among species but the main mechanisms of function are similar. In mammals, studies of PC2 are important for neurobiology and for understanding the mechanisms of obesity and diabetes. In invertebrates, it is established that PC2 and its potential substrates (neuropeptides and neurohormones) play important roles in early embryogenesis, egg-laying, hatching, etc. This makes the PC2 of H. glycines a good target protein for the development of novel anti-nematode compounds. Identification and analysis of the first proconvertase gene in plant parasitic nematodes should result in comprehensive studies of the direct role of the enzyme in nematode development and benefit the search for additional molecular targets in plant nematodes.
The architectural structure of HglPC2 has both typical and unique properties. The insert described here in the PC2 sequences of nematodes has not been reported before, and its biological significance remains unclear. This structural component appears to be not only nematode phylum-specific but also unique to type 2 proconvertases, since no other type of proconvertase possesses this feature. Preliminary analysis leads to speculations about the significance of the insert of plant nematode H. glycines in its relationship with the host plant, which requires further study.
Despite the high amino acid homology between the PC2s of two nematodes, we found that nucleotide and amino acid biases in HglPC2 are different from those observed in PC2 of C. elegans: the first is GC-rich and the latter is AT-rich. The nucleotide composition of most genes of nematodes is AT biased. This was shown for genome data from several free-living and animal parasitic nematodes and a limited number of plant nematode genes, primarily from Meloidogyne spp. To explore the contradictory data on GC richness of HglPC2, we analyzed PC2 fragments from other plant nematodes. The comparison showed the similar GC richness in G. rostochiensis, which belongs to the same subfamily (Heteroderinae), whereas a more distant relative of H. glycines, M. incognita, was even more AT biased than C. elegans. The same differences in base composition were observed for other gene sequences of these species.
There are three main outcomes from these observations. First, the discovered GC bias in PC2 of H. glycines and G. rostochiensis presents an exception to the general AT richness of nematode genes, including the plant nematode Meloidogyne spp. Since H. glycines and G. rostochiensis are members of the same subfamily, the interesting question of the distribution of GC richness among plant nematode species is raised. Second, base composition phylogenetics, apart from amino acid homology of proteins, may help to resolve some of the inconsistencies and complexities in current nematode classification. Such complexity was addressed by a recent hypothesis about evolutionary relationships among free-living animal and plant nematodes. Third, there is practical application of the data. Differences between the GC content of sequences from the model nematode C. elegans and those from H. glycines are great enough that data derived from C. elegans are not effective in primer design or predicting codon usage pattern. In our study, sequences from GC-rich D. melanogaster PC2 and the farthest relative human PC2, which surprisingly had the closest codon indices values to H. glycines, were very helpful in designing probes for screening the H. glycines library. The data presented here should therefore be useful in the design of molecular probes, minimizing the degeneracy of PCR primers for other plant nematode genes in cases where sequences from closely related species are not known.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0940fje; to cite this article, use FASEB J. (May 8, 2002) 10.1096/fj.01-0940fje. 
2 Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
