Molecular Biology Reports 30: 239–242, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.

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Characterization of a novel splicing variant of KLHL5∗ , a member of the kelch protein family Jian Xu1,
, Shaohua Gu1,
, Shu Wang1 , Jianliang Dai1 , Chaoneng Ji1 , Yangsheng Jin2 , Ji Qian1 , Liu Wang1 , Xin Ye1 , Yi Xie1,∗∗ & Yumin Mao1,∗∗ 1 State

Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, People’s Republic of China; 2 United Gene Holdings, Ltd, Shanghai, 200092, P.R. China

Accepted 16 April 2003

Key words: BTB/Kelch, kelch-repeat, KLHL5

Abstract The kelch-repeat protein family is a recently found new kind of actin-binding protein. It is characterized by tandemly arranged motifs of about 50 amino acids [7]. Previous study showed that most members of the kelch-repeat family were cytoskeletal proteins implicated in various cellular processes, such as actin cytoskeleton interaction, cytoplasmic sequestration of transcription factors and cell morphology [1]. And some of the family members play important roles in tissue development, such as human ENC-1, NRP/B, etc. Another characteristic of the kelch family is that most members have another conserved BTB domain at the extreme amino terminus. The BTB domain is also found at the N-terminus of 5–10% of zinc-finger transcription factor types and is a conserved protein-protein interaction motif [15]. During the large-scale sequencing analysis of a human fetal brain cDNA library we found a novel kelch-like protein gene 5, KLHL5 [11], KLHL5 has high identity with Drosophila kelch protein and many other family members. Here we report a novel splicing variants of KLHL5, named KLHL5b and the expression pattern of KLHL5b in many tissues. Abbreviations: kb – kilobase(s) or 1000 bp; aa – amino acid; kDa – kilodaltons. Introduction The actin-based cytoskeleton plays an important role in defining cell shape and morphology and in orchestrating many of the dynamic aspects of cell behavior such as cell migration, axon guidance, and cytokinesis. It also responsible for the generation and maintenance of cell polarity [8] and regulates organelle and protein distribution as well as mRNA transport between the nucleus and cytoplasm [2]. It is found that the assembly and disassembly of actin filaments are regulated by a number of actin-binding proteins ∗ The nucleotide sequence reported in this paper has been submitted to GenBank under accession number AY172948.
These two authors contributed equally to this paper. ∗∗ Corresponding authors: Yumin Mao, Tel: +86-21-65643573, Fax: +86-21-65642502, Email address: [email protected], Yi Xie, Tel: +86-21-55520025.

(ABPs). The kelch-repeat protein family is a recently found new kind of actin-binding protein. It is characterized by tandemly arranged motifs of about 50 amino acids [7]. The motif was first described as a distinct structural motif and termed the kelch motif in the Drosophila kelch protein [14]. Since then, members of the kelch family of proteins have been identified in several species from yeast to human. The kelch-repeat domain functions as a novel actin-binding protein. Direct evidence came from Limulus α-scruin, human Ipp and mayven in which the actin binding activity of the protein has been located specifically to the kelch domains [5, 7, 9]. In addition to the actin-binding activity, the kelch-repeat proteins have various functions in vivo. Human calicin is located within an actin-negative structure and is suggested an important role in spermatocyte differentiation [10]. NS1-BP (NS1-binding protein) was reported to interact with NS1 protein and

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Figure 2. Alignment of KLHL5b and KLHL5a. The conserved amino acids (GG-Y-W) of kelch-repeat family are indicated by black shade. Bold italics type shows the different BTB domains of the two variants.

Figure 1. The nucleotide and deduced amino acid sequences of KLHL5b gene. The cloned cDNA gave one extensive reading frame coding for a protein of 694 amino acids. The start and stop codons bordering the open reading frame are marked in bold italics type. Possible polyadenylation signals are underlined. Dark shading highlights the six well-conserved kelch motif and the BTB domain is indicated by black box. The sequence data reported here have been deposited in the Genbank (accession No. AY172948).

relocalized in the nuclei of infected cells, suggesting it had a role in mediating the splicing-inhibitory effect of the NS1 protein [12]. The crystal structure of kelch motifs in fungal galactose oxidase has been determined. Each kelch motif forms a four-strandedβ-sheet and the kelch motifs are predicted to assemble in a βpropeller structure [1, 4]. The β-propeller molecular module favors the coordination of multiple proteinprotein interactions including actin-binding activity. Another characteristic of the kelch family is that most members such kelch, calicin, NS1-BP, NRP-B have another conserved BTB domain at the extreme amino terminus. The BTB domain is also found at the Nterminus of 5–10% of zinc-finger transcription factor types and is a conserved protein-protein interaction motif [15]. The BTB domains could form homodimers or heterdimers thus amplified or modulate their functions. During the large-scale sequencing analysis of a human fetal brain cDNA library constructed by our

241 RT-PCR

Figure 3. Genome structure and alternative splicing of the human KLHL5 gene, and the exon composition of the two splicing variants. The cDNAs are from genome contig NT_016297.11. Filled boxes indicated coding regions while open boxes denote untranslated regions.

Figure 4. Expression pattern of KLHL5 gene in 16 tissues. RT-PCR analysis of KLHL5 and G3PDH (as a control) were performed as described in the text. Lane 1 heart; 2 brain; 3 placenta; 4 lung; 5 liver; 6 skeletal muscle; 7 kidney; 8 pancreas; 9 spleen; 10 thymus; 11 prostate; 12 testis; 13 ovary; 14 small intestine; 15 colon; 16 peripheral blood leukocyte.

laboratory [13], we isolated two cDNA clones with complete coding sequences, encoding two novel proteins, which show 43% and 40% identities with kelch of the kelch-repeat domain, respectively. Sequence analysis revealed these two cDNA clones are two different splicing variants of a novel kelch-like gene 5 (KLHL5), named KLHL5a and KLHL5b, respectively.

Two human Multiple Tissue cDNA (MTC) panels (CLONTECH) were used as PCR templates according to the manufacturer’s protocol. The sense primer sequence for KLHL5 was 5 -ATGGCATCCACCTCTG AAGTCCCT-3 , located at 100 bp upstream from the 3 terminus of the first exon and the antisense primer for KLHL5 was 5 -CAAGGCGGCCTGTATGAGCAT ACTGGA-3 , located at 100 bp downstream from 5 terminus of the third exon. G3PDH-specific primers were 5 -TGAAGGTCGGAGTCAACGGATTTGGT3 (G3PDHF) and 5 -CATGTGGGCCATGAGGTCC ACCAC-3 (G3PDHR). 24 cycles(for G3PDH) and 36 cycles (for KLHL5) of amplification (30 s at 94 ◦ C,30 s at 68 ◦ C and 1.0 min at 68 ◦ C) were performed using ELONGASE DNA polymerase(GIBCO BRL) . The PCR products of KLHL5a, KLHL5b and G3PDH were then electrophoresed on a 2% agarose gel.

Results and discussion The KLHL5 cDNA sequence During the large-scale sequencing analysis of a human fetal brain cDNA library constructed by our laboratory [13], we isolated two different splicing variants of a novel kelch-like gene 5 (KLHL5), named KLHL5a and KLHL5b, respectively. Sequence characterization

Material and methods cDNA library construction A cDNA library was constructed in a modified pBluescript II SK(+) vector with the human fetal brain mRNA purchased from CLONTECH [13]. Bioinformatics analysis DNA and protein sequence comparisons were carried out using BLAST2.0 at NCBI (http://www.ncbi.nlm. nih.gov/blast). Motif analysis was done with PROFILESCAN (http://www.isrec.isb-sib.ch /software/ PFSCAN_form.html). Other sequence analysis was performed by GCG (Wisconsin Package, Version 10.0) or online.

The cDNA of KLHL5a is 3488 bp long and contains an ORF of 2268 bp encoding a putative protein of 755aa [11]. While the cDNA of KLHL5b is 3305 bp long and contains an ORF of 2085 bp encoding a putative protein of 694aa with the molecular mass of 77.4 KD and a pI of 6.1 (Antheprot 5.0, Figure 1). PSORT II (http://srs.pku.edu.cn: 8088) predicted that both KLHL5a and KLHL5b are cytoplasmic proteins without N-terminal signal peptides. By scanning the deduced amino acid sequence against the PROSITE database (http://www.expasy.ch/tools/scanprosite), we found that both KLHL5a and KLHL5b have six wellconserved kelch-repeat motifs and one conserved BTB domain (Figure 1). Alignment of the kelch-repeat regions of KLHL5a, KLHL5b and kelch revealed a conserved pattern of residues, including a GlycineGlycine doublet that is an invariant feature in nearly all kelch repeats (Figure 2).

242 To determine the chromosome location of the KLHL5, the cDNA sequences were searched to the human genome database with the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST). The gene is mapped to contig NT_016297.11, from chromosome 4p14 spanning about 59.8 kb. The search of human EST database also identified multiple human ESTs that corresponded to KLHL5, grouped as the UniGene cluster Hs.272239. Comparing the cDNAs and genome sequence of KLHL5 revealed that the gene has 11 exons and 10 introns. KLHL5a is made up of all the 11 exons while KLHL5b misses the second exon (Figure 3). So KLHL5b is composed with 10 exons and 9 introns and is 183 bp shorter than KLHL5, thus the deduced KLHL5b protein is 61 amino acids shorter than that of KLHL5a. The absence of the 61 amino acids occurs within the BTB domain of KLHL5b, which may truncate part of the ‘core region’ of the BTB domain of KLHL5b (Figure 2). We speculate that the functions mediated by the two BTB domains in vivo may be different since the truncation of BTB domain of KLHL5b may prevent the proper folding of the domain. Both sequences of the exon-intron junctions are consistent with the AG-GT rule (data not shown). Expression pattern of KLHL5a and KLHL5b In order to investigate the tissue expression pattern of the two splicing variants, we performed RT-PCR. The result showed that both KLHL5a and KLHL5b were expressed ubiquitously in the sixteen tissues examined (Figure 4). They were strongly expressed in adult heart, brain, placenta, liver, kidney and pancreas, while weakly in lung, spleen, ovary and small intestine. What interested us most was that the expression patterns of KLHL5a and KLHL5b were different in kidney and pancreas. So we speculated that the different tissue expression pattern of KHLH5a and KHLH5b might imply various functions. In this paper, we reported a novel splicing variant of KHLH5 named KLHL5b. The alternative splicing caused the BTB domains of the variants different

from each other. Previous study has demonstrated that BTB domain may participate in a variety of processes ranging from transcriptional regulation to cytoskeleton organization [3]. The altered BTB domain of KLHL5b may regulate the function of KLHL5a via homo/heterdimerization or multimerization. Further study may focus on elucidating the exact functions of BTB domain and kelch domain, and looking for the relations between some phenomenon and these two splicing forms.

Acknowledgements This research is a part of Project 30170345 supported by the National Natural Science Foundation of China.

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