Sequence Analysis of GDSL Lipase Gene Family in Arabidopsis
To analyze sequence characters of GDSL lipase gene
family in Arabidopsis thaliana, 108 members of GDSL lipases were
analyzed using data mining. The gene structures display remarkable diversity,
consisting of zero to 13 introns. And the genes are asymmetrically distributed
in chromosome 1-5, some of which are arranged in tandem. Phylogenetically,
they were classified into three groups. Lipase-GDSL domain (PF00478) is
housed at or close to N-terminus, or in the middle of amino acid sequences,
additionally in which other domains and replicates were also found. Most
GDSL lipases contain a signal peptide for conducting the secretary pathway.
They are predicted to be extracellularly secreted, or target to mitochondria,
chloroplast or any other parts of the cells. Functionally, these lipases
are potentially involved in multiple physiological roles including seed
germination, flowering and defense reactions. This study will help further
understand the sequences and functions of Arabidopsis GDSL lipases.
GDSL lipases widely exist in microbe and plant species.
As an important gene family of lipases, GDSL lipases are active in hydrolysis
and synthesis of lipids or esters. A serine-containing GDSL-motif close
to the N-terminus, five conservative blocks and a Ser-Asp-His triad are
included in the ammonic acid sequences (Upton and Buckley, 1995). GDSL
lipase is generally made up of several ß-strands and a-helices arranged
in alternate order and the substrate-binding pocket between the central
ß-strand and long a-helix appears to be highly flexible. The flexible
pocket brings conformational changes, so that the active sites exposure
to the solvent and easily bind to substantive substrates. Possessing multiple
functions, GDSL lipases are potentially applied to the food, flavor, fragrance,
cosmetics, textile, pharmaceutical and detergent industry (Akoh et
al., 2004; Ling et al., 2006).
Physiologically, plant GDSL lipases are generally considered
to be mainly involved in the regulation of plant growth and development.
Recently the research in this field becomes more and more attractive.
Although several candidates from A. thaliana, Rauvolfia serpentina,
Medicago, Hevea brasiliensis, Alopecurus myosuroides have been extracted,
cloned and characterized (Brick et al., 1995; Oh et al.,
2005; Ruppert et al., 2005; Pringle and Dickstein, 2004; Arif et
al., 2004; Cummins and Edwards, 2004), such understanding of GDSL
lipases is still limited. Comparatively, complete sequencing of the Arabidopsis
genome accelerates cloning and characterization of lipases and information
of Arabidopsis GDSL lipases deposited in the databases is rich
and detailed. However, identification and functional analysis of them
in databases or literatures is not enough or incomplete. Although a small
number of Arabidopsis GDSL lipases (designated as AGLs) have been
phylogenectically analyzed and aligned by Akoh et al. (2004), further
comprehensive information is required by performing gene structure, multiple
alignment, conserved motif or domain search and analysis of subcellular
MATERIALS AND METHODS
This study was conducted at Zhengzhou (China) in the year
The original set of the AGL cDNAs was searched at
the Arabidopsis Information Resource [http://www.arabidopsis.org],
then compiled and translated into amino acid sequences by Vector NTI Suite
The other protein sequences were retrieved from GenBank http://www.ncbi.nlm.nih.gov/Genbank/index.html]
and EBI databases [http://www.ebi.ac.uk/interpro/].
All the gene structures were analyzed in website [http://www.ncbi.nlm.
nih.gov/Entrez/query.fcgi?db=gene]. The motifs or domains of AGL proteins
were analyzed using InterProScan [http://www.ebi.ac.uk/InterProScan/].
Sequences without GDSL-lipase domain or motif were removed. The left AGL
protein sequences were aligned using Vector NTI Suite 8.0 to construct
representative sequences. The gene loci distribution was performed by
Chromosome Map Tool (http://www.arabidopsis.org/jsp/ChromosomeMap/tool.jsp).
The phylogenetic analysis was carried out using the Neighbor-Joining method
with MEGA3 http://www.megasoftware.net/].
Analysis of subcellular localization was performed for all the left AGL
protein sequences by the target P program version 1.1 http://www.cbs.dtu.dk/services/TargetP].
RESULTS AND DISCUSSION
Molecular analysis of multiple GDSL lipases and genes:
Data mining was used to search all the cDNA sequences encoding
AGLs. Using NCBI and TAIR database searches, 121 AGL cDNA sequences
were obtained (data not shown). After sequence compilation, translation
and alignment, thirteen cDNAs encoding other proteins were removed. Because
two sequences (Acc. No. NM_118813 and NM_179120) are translated into one
protein, the 108 cDNAs corresponding to 99 Atg loci putatively encode
107 GDSL lipase proteins.
As a result, two genes (Acc. No. NM_118813 and NM_179120)
that encode one protein have different gene structures, so the structure
and organization analysis of total 108 genes was performed in website.
It indicated that the gene structures showed remarkable diversity. And
it is likely that all of them except for one gene (Acc. No. AY058847)
contain different numbers of introns. Of the analyzed genes, 73 genes
consist of four introns; 11 and 12 have three and two introns, respectively.
One gene (Acc. No. NM_101867) containing 13 introns is the most complicated
and predicted to encode a high molecular weight protein of 1006 amino
acid residues, while another one (Acc. No. NM_113550) with eight introns
putatively encodes a much smaller protein of only 380 amino acids. And
the other nine genes contain one or five introns. These introns and exons
have the length of several decades of base pairs to over one kilo-base
Analyzed by Chromosome Map Tool, GDSL lipase genes are distributed
in all chromosomes although not uniformly (Fig.
1). Forty seven genes exist in Chromosome 1 and 23 genes are distributed
in Chromosome 5. Fourteen and Fifteen genes exist in Chromosome 2 and
3, respectively. Nevertheless, only eight genes are located in Chromosome
4. There are some regions with a high density of genes, such as the middle
and bottom of Chromosome 1, the top and bottom of Chromosome 5. Furthermore,
there are 12 cases of two or more genes arranged in tandem. For example,
six genes (Acc. No. NM_202420, NM_106239-NM_106243) that encode extracellular
lipases (EXL1-6, respectively) are arranged in tandem at the bottom of
Chromosome 1, which is consistent with the previous study (Mayfield et
al., 2001). In addition, some of them are likely duplicated in the
areas of inter- or intra-chromosome, resulting in multiple gene copies
The obtained cDNA sequences were putatively translated into
107 amino acid sequences, consisting of 118-1006 amino acid residues,
with the molecular weight from 12.9-109.1 kDa. Five blocks (block 1-5)
containing the conserved sequences (PAIFVFGDSIVDTGNNN, TGRFSNGRLIXD, ALYLIXIGXNDY,
LYXLGXARK XXVXGLXXXPLGCLP and YVFWXDXXHPTEXA, respectively) were determined
by aligning the obtained sequences. Combining the previous reports (Oh
et al., 2005; Akoh et al., 2004), we predict that the active
serine, aspartate and histidine (in block 1 and 5, respectively) likely
constitute the active triad.
Phylogenetic relationship was analyzed by the method of
neighbor-joining. They were classified into three groups (Group 1, 2 and
2), composed of 28, 40 and 39 members, respectively. Proteins designated
as APG (Acc. No. AAL24235) and EXL1-6 were included in Group 2 and reported
to be involved in flowering (Upton and Buckley, 1995; Mayfield et al.,
2001) and GLIP1 (Acc. No. NP_198915, encoded by At5g40990) in Group 2
was involved in defense against the necrotrophic fungus Alternaria
brassicicola as shown by Oh et al. (2005). Additionally, Arab-1
(Acc. No. NP_174188) belongs to Group 3.
Domain/motif structures of GDSL lipase proteins: Analysis by InterProScan
indicated that all of the 107 proteins contained lipase-GDSL domain (PF00657).
Whereas its location, number and kind are different, which leads to five
classes of AGLs (class A-E) detailed in Table 1. It reveals
that most AGLs in class A contain only lipase-GDSL domain close to N-terminus,
which is consistent with characteristics of traditional GDSL lipases.
Several to tens of amino acid residues in front of the lipase-GDSL domain
likely shape into a signal peptide (SP). However, lipase-GDSL domain is
located at the N-terminus and in the middle of two proteins (Acc. No.
NP_196001 and AAL24235), respectively. Interestingly, three lipase-GDSL
domains were found in NP_173441. Furthermore, one protein (Acc. No. NP_177718)
contains two different domains (lipase-GDSL domain and 5`-nucleotidase/apyrase
domain), suggesting that it might function as a GDSL lipase and a nucleotidase/apyrase.
Although exist the above-mentioned differences, domain/motif structure
of AGLs is comparatively uniform.
domains located in AGL proteins
L: Lipase-GDSL domain, NA:
5`-nucleotidase/apyrase domain, AGL: Arabidopsis GDSL lipase
Without enough resources of three-dimensional structure
of plant GDSL lipases in the databases, presently three-dimensional structure
could not be performed by SWISS-MODEL server.
Functionally, these lipases are potentially involved in multiple physiological
roles. First of all, some play an important role in flowering. The anther-specific
proline-rich protein (Acc. No. AAL24235) and extracellular lipases (EXL1-6)
found in pollen coat have been identified (Brick et al., 1995;
Mayfield et al., 2001). Secondly, some GDSL lipases function in
disease resistance. For example, GLIP1 appears to trigger systematic resistance
Phylogenetic analysis of Arabidopsis
GDSL lipases the phylogenetic relationship of Arabidopsis GDSL
lipase proteins were analyzed using the Neighbor-Joining method
in plant species when challenged by A. brassicicola
(Oh et al., 2005). Additionally, other GDSL lipases potentially
take roles in seed germination and other issues as well. This kind of
protein in post-germinated sunflower (H. annuus L.) seeds shows
fatty acid-ester hydrolase activity (Beisson et al., 1997). And
an acetylajmalan esterase (designated as AAE, homologous to protein NP_174181)
from Rauvolfi plays an essential role in the late stage of ajmaline
biosynthesis (Ruppert et al., 2005). However, further evidences
are required to support such predictions.
Prediction of subcellular localization: It reveals that 99 proteins
in type 1 contain signal peptide, putatively involved in targeting to
endoplasmic reticulum or subsequent transport through secretory pathway
(Table 2). The signal peptides consist of 16 to 113 amino
acid residues. A signal peptide was found in Arab-1 (Acc. No. NP_174188)
and EXL1-6. Other four proteins (Acc. No. NP_199404, NP_201098, NP_199004
and NP_196001) were predicted to target to any parts in cells. Two proteins
(Acc. No. NP_195980 and NP_173441) potentially target to mitochondria
and chloroplast, respectively. While the subcellular localization of APG
protein (Acc. No. AAL24235, also P40602) is still unclear. In Arabidopsis,
a subfamily of genes encoding six extracellular lipases (EXL1-6) from
pollen coat has been reported (Mayfield et al., 2001). Hereby,
in addition to being extracellularly secreted, GDSL lipases with signal
peptide probably target to different organelles or subcellular parts.
localization prediction of AGL proteins
In conclusion, we analyzed the Arabidopsis GDSL lipase
gene family of 108 members distributed in chromosome 1-5 and putatively
encoding 107 GDSL lipase proteins. The gene structures, phylogenetic relationship,
domain/motif organization and subcellular localization have been performed.
It`s a superfamily of putative GDSL lipase genes potentially playing important
roles in regulation of Arabidopsis growth and development, including
seed germination, flowering and defense reactions. In the future, further
data of precise subcellular localization, three-dimensional structure
modeling, mutagenesis, over-expression/recombinant expression, substrate
selectivity will be done to understand more about Arabidopsis GDSL
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