Http://www.abbs.info e-mail:[email protected] ISSN 0582-9879 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2001, 33(1): 19-24 CN 31-1300/Q |
Molecular
Cloning of NELIN, a Putative Human Cytoskeleton Regulation Gene
( Molecular Medicine Center for Cardiovascular
Diseases, Fu Wai Heart Hospital & Cardiovascular Institute,
FAs
are large integrins aggregates found at the ends of prominent bundles of actin
filaments (namely stress fibers). During the formation of a stable interaction
between a cell and its ECM, integrins become clustered resulting in the
accumulation of cytoplasm proteins (such as cytoskeleton proteins and a large
varieties of associated proteins, adaptors, protein kinases and etc.) to the
cytoplasmic domains of the integrins, where focal adhesion forms. Concurrent
with these events, filaments of the actin-based cytoskeleton coalesce into
progressively thicker bundles to form the stress fibers and interacts with
integrins. The subsequent cytoskeletal and signaling complex can initiate
several down-stream signaling pathways, which regulate functions of cells or
determine the fate of cells[4].
Large-scale
ESTs sequencing become available since fantastic spurt in autosequencing and
bioinformatics. As an efficient way, it plays indispensable roles in hunting
for novel genes and towards understanding the expression profiles of human
genes[5]. For searching cardiovascular-associated genes and
investigating their expression profiles, we have constructed human adult heart
and aorta cDNA libraries, and isolated several novel genes with putative
important functions based on large-scale ESTs sequencing[6]. Here,
we report the isolation of a novel gene NELIN from adult heart cDNA
library, which probably is muscle-specific(clone No. H409). Based on framework
of domain, NELIN can regulate the formation of stress fibers, focal adhesion
and its signaling complex, and probably participates in the signal transduction
in FAs.
1 Materials
and Methods
1.1 Large-scale ESTs sequencing from the
human adult heart cDNA library
The
cDNA library plating and subsequent large-scale sequencing have been recently
described[6].
1.2 Rescue and full length nucleotide
sequencing of H409
The
insert of H409 clone was excised in vivo from the ZAP express vector
using the ExAssist/XLOLR helper phage system (Stratagene), and DNA was purified
using alkaline lysis miniprep[7]. H409 cDNA clone was sequenced
using Taq Dyedeoxy Terminator Cycle Sequencing kit for Applied
Biosystems 377 Sequencing System (Perkin Elmer).
1.3 Full-length cloning and computer
analysis of H409
A
clone H409 exhibiting sequence similarity to autoantigen gene of human thyroid associated
ophthalmopathy patient (partial coding regions, GenBank accession number
S67069) was identified during the course of ESTs sequencing. Nucleotide
searches were performed using BLAST[8] against the GenBank (Release
116.0)/EMBL (Release 62)/DDBJ (Release 40), protein against SWISS-PROT (Release
38.0) and TrEMBL(31-Mar-2000). And further analyses after the complete coding
regions was obtained were performed just as follow: Protein domains against
PROSITE(Release 16) at ExPASy[9] and Pfam (version 3.1) at Sanger
center[10]. PCR primer design and related amino acid sequences
analysis were with the aids of Vector NTITM Suite (Informax, Inc).
1.4 Northern blot and RT-PCR
Multiple human tissue Northern blot (Clontech) was hybridized with the cDNA probe corresponding to the full-length of NELIN according to manufacture's instruction. The probe was labeled by a random priming using Prime-a-Gene Labeling System (Promega). Total RNA in heart, skeletal muscle, liver, kidney and lung were extracted from the spontaneously aborted fetus of 8-month. The transfer of total RNA to the nylon membrane filters(Schleicher & Schuell) and subsequent procedure was as described[10]. In each lane, 30 mg total RNA were determined by UV spectrometry. For determining the tissue-specific distribution of NELIN, total RNA were prepared from various human adult tissues, ie. artery, vein, brain, heart, liver, spleen, lung and kidney, and reverse transcription-polymerase chain reaction (RT-PCR) was performed. The primers were as follows: 5' primer, CAA CGA CCA TCT CTC AAG GA; 3' primer, GTA GTC ATC AGT TTC AAT GG.
1.5 Chromosomal localization analysis
Novel
genes could be assigned to chromosome directly by database analysis for the
sake of the completion of the human genome transcript map. The chromosomal
localization of NELIN was achieved by searching Uni.Gene database and
GeneMap’99[11,12].
2 Results
2.1 The full length of NELIN and
analysis of deduced amino acid sequences
The
nucleotide and predicted amino acid sequences of NELIN are shown in
Fig.1.
Fig.1 cDNA and predicted amino
acid sequences of NELIN
NLS
is in shade. Polyadenylation signal is underlined. Stop codon is indicated by
asterisk (*).
Fig.2 Comparison of F-actin
domain of NELIN with nexilin
The
range indicates the position of the domain within each protein. Shading
indicates identical residues.
Fig.3 Multiple alignment of
F-actin domain of NELIN with MYPC
The
alignment was constructed using Clustal W program[14]. Accession
number for aligned sequences follow: NP_032679, X84075, U38949, Q00872.
Multiple
fetal and adult tissue Northern blot have shown: (1) NELIN was detected
in heart and skeletal muscle only. At least two alternative splicing
transcripts i.e. 2.7 and 4.0 kb were observed in adult heart. The redundances
in order of abundance were: 4.0, 2.7 kb transcripts. And 2.7 kb transcript is
corresponding to the clone we had isolated. NELIN was expressed as 4.0
kb transcript in skeletal muscle(Fig.4). (2) Development-specific pattern: To
estimate mRNA levels, the densities of bands on autoradiograms were quantified
with the optical scanner(GS-700 Densitometer, Bio-Rad) and cor-rected to b-actin
mRNA. The expression level of 4.0 transcript in fetal heart was 3.4-fold
compared with skeletal muscle and decreased to 0.91-fold in adult. And 2.7 kb transcript
was rarely expressed in fetal heart.
Fig.4 Expression pattern of
human NELIN in fetal tissues (A) and in adult tissues (B)
(A) 1, kidney; 2, liver; 3, lung; 4, brain;
5, heart; 6, skeletal muscle. (B) 1, heart; 2, brain; 3, placenta; 4, lung; 5,
liver; 6, skeletal muscle; 7, kidney; 8, pancreas. Upper panel: The positions
of 28 S, 18 S and 5 S ribosomal RNA or RNA marker are indicated at left. Bottom
panel: As a control, the same blot was rehybridized with a radiolabeled b-actin
cDNA to confirm integrity and equal loading of mRNA from different tissues.
Fig.5
RT-PCR analysis of NELIN in adult tissue
1, artery; 2, vein; 3, brain; 4, heart;
5, liver; 6, spleen; 7, lung; 8, kidney; 9, control; M, 100 bp DNA ladder
(Promega). The PCR product of NELIN is 1386 bp, and β-actin product is
201 bp.
NELIN
has been included into the HS. 216381 in Uni.Gene database, which contains
marker stSG30971. A search of GeneMap'99 showed it is localized in interval
D1S203-D1S2865 on chromosome 1. Further analysis of chromosome 1 comprehensive
map in Rockefeller university[15] indicated that the genomic
sequence of NELIN could be localized between chromosome 1p31 and 1p32. A
log-odds (LOD) score of p1.83 indicated the confidence for the marker
stSG30971.
Integrin-mediated
adhesion triggers assembly of stress fibers. When bound to ECM ligands,
integrins become clustered and associated with focal complexes, triggering
assembly of stress fibers. That actin cytoskeleton assembles into stress fibers
can promote clustering of integrins further and make integrins bind to ECM more
effectively. That's bidirectional signaling between the integrins and
cytoskeleton[16]. Formation of FAs and assembly of stress fibers are
governed by Rho family which is subject to ras superfamily[17].
Recent studies have found that as cross-linkers between the actin cytoskeleton
and plasma membrane, N-terminals of ERM(ezrin, radixin and moesin) proteins
attach to the membrane through FERM domain and C-terminals interact with
filamentous actin (F-actin) cytoskeleton. ERM family behaves as regulatable
scaffold proteins that anchore actin filaments to the membrane, which is an
essential prerequisite for Rho to induce stress fibers. It seems that ERM
proteins are emerging as key regulators of the actin cytoskeleton and essential
for Rho-induced cytoskeletal effects. As an important signal transducer, ezrin
can also convey an antiapoptotic signal through the phosphatidylinositol
3-kinase/Akt pathway[18].
During
our analysis of NELIN, Ohtsuka T and coworkers cloned the b-, s-nexilin
genes, which are 96% similar to NELIN. S-nexilin is localized at the
ends of stress fibers, specifically at focal contacts but not at cell-cell
adherens junction, which supports the idea that they can anchor stress fiber to
the membrane through FERM-like domain[19]. Comparison with ERM
proteins, NELIN perhaps possesses myosin binding activity besides F-actin
binding activity. Although there is no sequence homology between the primary
structures of NELIN and ERIN, the framework of domain suggests they can share
similar roles.
Cardiac
myocytes not only have focal adhesions, but also form cytosketal-sarcolemmal
attachments (i.e. costameres) in register with their Z lines. Costameres are
similar to focal adhesions in many aspects. NELIN probably regulates the
formation of costameres in regard to its framework of domain and homologous
proteins. Previous studies have demonstrated that mechanical load is the major
cause of cardiac hypertrophy. However, the mechanisms how mechanical force is
transduced into biochemical signals remain unclear[20]. It seems
that FAs and costameres are the starting site for mechanical tension
transduction. If they do serve as mechanoreceptor to transduce mechanical
signal into biochemical ones, then alteration in mechanical load would greatly
affect the size, biochemical composition, location of FAs and costameres, and
initiate the down stream signals to cause cardiac hypertrophy.
Northern
blot and RT-PCR results have demonstrated that NELIN is only present in
heart, skeletal muscle, artery and vein, so it seems reasonable to be concluded
that NELIN is muscle-specific(cardiac myocytes, skeletal muscle and
smooth muscle), perhaps 2.7 kb transcript is cardiac-specific and therefore may
play more specific roles in cardiac myocytes. It's intriguing that 2.7 kb
transcript is expressed differentially in heart in addition to 4.0 kb in
skeletal muscle, and given that many cytoskeleton regulation proteins have
shown to be important signal tranducers and NELIN contains NLS sequences; Taken
together, we've postulated that NELIN translocates from cytoplasma to the
nucleus and interacts with transcription factors to regulate the transcription
of target genes in integrins signaling pathways. It remains to be determined
whether and how NELIN involves in the development of heart.
It's
puzzled that NELIN shares high homology with autoantigen gene. Perhaps
IG domain of NELIN is recognized by autoimmune system, which would cause
autoimmune disorder. Further studies will be needed to elucidate the exact role
of NELIN in the process.
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Received: June 30, 2000 Accepted: September 4, 2000
This work was supported by a grant from
Ninth Five Years Plan of National Medical Science and Technique
Foundation(No.96-906-02-07); The nucleotide sequences reported in this paper
have been submitted to the GenBank database under the accession number AF114264
*Corresponding author:Tel,
86-10-68314466-8003 or 86-10-68332561; Fax, 86-10-68313012; e-mail,
[email protected]