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

ZHAO Yong, WEI Ying-Jie, CAO Hui-Qing, DING Jin-Feng*

( Molecular Medicine Center for Cardiovascular Diseases, Fu Wai Heart Hospital & Cardiovascular Institute, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100037, China )

 

Abstract        For searching cardiovascular-associated genes and investigating their expression profiles, human adult heart and aorta cDNA libraries were constructed, and a novel gene from adult heart cDNA library was isolated based on large-scale ESTs (expressed sequence tags) sequencing (GenBank accession number AF114264). The 2736 bp clone contains one 1 344 bp open reading frame extending from 412 to 1755. We named it NELIN (nexilin-like protein) because it shares high similarity with the rat nexilin. NELIN was expression-restricted in heart, skeletal muscle, artery and vein by Northern blot and RT-PCR analyses, and mapped to chromosome 1p31-1p32 by database analyses. Based on domain structure, NELIN could regulate the formations of stress fibers, focal adhesion and its signaling complex, and even participates in the signal transduction in FAs (focal adhesions).

Key words    molecular cloning; cytoskeleton regulation gene; focal adhesions; signal transduction; muscle-specific

 

Integrins and FAs(focal adhesions or focal contacts) play critical roles in almost all the eukaryotic cells. FAs and its complexes become attractive in recent years for they represent signaling hot spots. However, the underlying mechanisms of their signaling and regulation remain unclear. Cells adhesion to ECM(extracellular matrix) are extremely important in directing the migration, proliferation and differentiation of the cells; especially essential for continuous contractile activity of heart and adaptive to environmental stress(e.g. hypoxia), aberrations in such interactions can lead to pathological disorders. As we know, integrins function as: (1) regulation cell cycles; (2) control of cell shape, growth and survival; (3) integrin signaling pathways synergize with other receptors, such as RTKs, GPCRs, cytokines and immune response receptors and etc. Undoubtedly, cross talk between integrins and other receptor pathways is critically involved in the integration of signals that converged on cells in their natural environments in vivo[1-3].

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 (*).

 

Bioinformatics analyses indicated that the amino-terminal contains a putative FERIN-like domain which can possibly attach to the membrane. A F-actin binding domain which shares high similarity with the rat b, s-nexilin locates on 76-242 of the amino acid sequences (Fig.2). IG domain in the carboxy-terminal bears 50% similarity to the counterpart of MYPC, which can bind to myosin (Fig.3)[13].

 

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.

 

2.2 The tissue and development-specific distribution of NELIN

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.

 

RT-PCR results showed that NELIN is expressed in cardiac myocytes, artery and vein(Fig.5).

 

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.

 

2.3 Chromosomal assignment

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.

3    Discussion

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.

 

References

1     Clark EA, Brugge JS. Integrins and signal transduction pathways: the road taken. Science, 1995, 268: 233239

2     Giancotti FG, Ruoslahti E. Integrin signaling. Science, 1999, 285: 10281032

3     Hsueh WA, Law RE, Do YS. Integrins, adhesion, and cardiac remodeling. Hypertension, 1998, 31: 176180

4     Schmidt A, Hall MN. Signaling to the actin cytoskeleton. Annu Rev Cell Dev Biol, 1998, 14: 305338

5     Liew CC, Hwang DM, Fung YW, Laurenssen C, Cukerman E, Tsui S, Lee C Y. A catalogue of genes in the cardiovascular system as identified by expressed sequence tags. Proc Natl Acad Sci USA, 1994, 91: 1064510649

6     Wei YJ, Ding JF, Zhao Y, Wang XR, Liu Y Q, Xu YY, Xiong H et al. Construction of human adult artery and heart cDNA libraries and cloning of novel full-length cDNAs. Chinese Circulation Journal, 1999, 14: 369371

7     Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd ed, Beijing: Science Press, 1992, 1922, 362369

8     Altschul SF, Madden TL, Schaffer AA. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res, 1997, 25: 33893402

9     Hofmann K, Bucher P, Falquet L, Bairoch A. The PROSITE database, its status in 1999. Nucleic Acids Res, 1999, 27: 215219

10    Sonnhammer EL, Eddy SR, Birney E, Bateman A, Durbin R. Pfam: Multiple sequence alignments and HMM-profiles of protein domains. Nucleic Acids Res, 1998, 26: 320322

11    http://www.ncbi.nlm.nih.gov/UniGene/

12    http://www.ncbi.nlm.nih.gov/genemap/

13    Winegrad S. Cardiac myosin binding protein C. Circ Res, 1999, 84: 11171126

14    Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res, 1994, 22: 46734680

15    http://linkage.rockefeller.edu/chr1

16    Schoenwaelder SM, Burridge K. Bidirectional signaling between the cytoskeleton and integrins. Curr Opin Cell Biol, 1999, 11: 274286

17    Nobes C D, Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell, 1995, 81: 5362

18    Gautreau A, Poullet P, Louvard D, Arpin M. Ezrin, a plasma membrane-microfilament linker, signals cell survival through the phosphatidylinositol 3-kinase/Akt pathway. Proc Natl Acad Sci USA, 1999, 96: 73007305

19    Ohtsuka T, Nakanishi H, Ikeda W, Satoh A, Momose Y, Nishioka H, Takai Y. Nexilin: A novel actin filament-binding protein localized at cell-matrix adherens junction. J Cell Biol, 1998, 143: 12271238

20    Sharp WW, Simpson DG, Borg TK, Samarel AM, Terracio L. Mechanical forces regulate focal adhesion and costamere assembly in cardiac myocytes. Am J Physiol, 1997, 273: H546556


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]