Introduction

Mental retardation (MR) is the most common developmental disability and ranks first among the chronic conditions which causing major activity limitations in the United States. A genetic or inherited metabolic etiology was implicated in two-thirds of mental retardation cases [1], and a recessive mode of transmission accounts for nearly one-fourth of these cases [2]. Several of these cases affected basic cellular mechanisms such as cell signaling pathways [3–6], regulation of gene expression [7–11], and alterations in hippocampal dendrite morphology [12]. Autosomal recessive nonsyndromic mental retardation (ARNSMR) which associated with IQs ranging from 50 to 70 was widely studied.

Higgins and colleagues found that the homozygous C > T nonsense mutation at nucleotide position 1,274 of a novel cDNA (1,274C > T) is involved in autosomal recessive nonsyndromic mental retardation (ARNSMR) in a large kindred [13]. The gene was named CRBN (cereblon, NM_016302) based on its putative role in cerebral development and the presence of its large, highly conserved Lon domain [13]. The nonsense mutation causing a premature stop codon in CRBN interrupts an N-myristoylation site and eliminates a casein kinase II phosphorylation site at the C terminus [13]. As casein kinase II is activated during introduction of long-term potentiation, these disruptions may prevent its proper subcellular targeting and alter long-term potentiation in the hippocampus [14]. As most human Lon-containing proteins are localized to the mitochondria where they selectively degrade short-lived polypeptides [15, 16], it is hypothesized that the mutation perturb nuclear regulation of mitochondrial energy metabolism and cause memory and learning difficulties [13]. Recent studies showed that the rat homolog of CRBN (rCRBN) might play an important role in assembly and surface expression of functional BKCa channels by direct interaction with the cytosolic C-terminus of its α-subunit [17]. However, studies on human CRBN protein are rare to date.

The predicted protein of CRBN, LON protease might belong to a family of adenosine triphosphate (ATP)-dependent protease which is highly conserved across species [18]. Energy-dependent proteolysis plays a key role in prokaryotic and eukaryotic cells by regulating the availability of certain short-lived regulatory proteins, ensuring the proper stoichiometry of multiprotein complexes, and ridding the cell of abnormal and damaged proteins [19]. Some ATP-dependent Lon proteases were reported to be associated with mental retardation. PRSS15, which encodes ATP-dependent Lon protease is highly expressed in the human brain. Although the PRSS15 gene had not been directly associated with human disease, its presence in the hippocampus [19] and its location in the candidate region for a severe form of ARNSMR [20] suggested a role in memory and learning.

During a large-scale sequencing analysis of a human fetal brain cDNA library, we isolated the full-length cDNA of CRBN. We analyzed its bioinformatics, gene expression pattern and subcellular localization to elucidate the putative roles of CRBN.

Materials and methods

cDNA library construction

The cDNA of CRBN gene was cloned from the human fetal library during large-scale cDNA sequencing. The cDNA library was constructed into a modified pBluescript II SK (+) vector with the human fetal brain mRNA (Clontech). The modified vector was constructed by introducing two SfiI recognition sites, i.e. SfiI A (5′-GGCCATTATGGCC-3′) and SfiI B (5′-GGCCGCCTCGGCC-3′), between the EcoRI and NotI sites of pBluescript II SK (+) (Stratagene). Double-stranded cDNAs were synthesized using SMART™ cDNA Library Construction Kit (Clontech) following the manufacturer’s instructions. The cDNA inserts were sequenced on an ABI PRISMk 377 DNA sequencer (Perkin-Elmer) using the BigDye Terminator Cycle Sequencing Kit and BigDye Primer Cycle Sequencing Kit (Perkin-Elmer) with 21M13 primer, M13Rev primer and synthetic internal walking-primers designed according to the obtained cDNA sequence fragments. Each part of the insert was sequenced at least three times bi-directionally. Subsequent editing and assembly of all the sequences from one clone was performed using Acembly (Sanger’s Center).

Sequence analysis

Chromosome localization of the human CRBN gene was determined by comparing the cDNA sequence with the human genomic sequences at NCBI (http://www.ncbi.nlm.nih.gov/genome/seq/BlastGen/BlastGen.cgi?taxid=9606). DNA and the putative protein sequence comparisons were carried out using the BLAST tool at NCBI (http://www.ncbi.nlm.nih.gov/BLAST). Multiple sequence alignment analysis was done with Genedoc software (http://www.psc.edu/biomed/genedoc).

Assessment of CRBN mRNA tissue distribution by RT-PCR

Human multiple tissue cDNA (MTC) panel was used as PCR templates according to the manufacturer’s protocol. All panels were purchased from Clontech. The primer sequences of the human CRBN gene were 5′-ATGGCCGGCGAAGGAGATCAG-3′ (CRBNf) and 5′-TTACAAGCAAAGTATTACTTTG-3′ (CRBNr). Twenty-four cycles (for G3PDH) or 36 cycles (for CRBN) of amplification (30 s at 94°C, 30 s at 68°C and 90 s at 72°C) were performed using ELONGASE DNA polymerase (INVITROGEN). PCR primers CRBNf and CRBNr are both located in the whole open reading frame of the CRBN gene, spanning 1329 base pairs. The PCR products of CRBN and G3PDH were then electrophoresed on a 1% agarose gel.

Subcellular localization of CRBN and CRBN′

CRBN and CRBN′ (the mutation of CRBN at 1274 site) were amplified by PCR. The primer sequences were 5′-CCGCTCGAGTGGCCGGCGAAGGAGATCAGCAGGACG-3′ (CRBN-P1), 5′-CGGGATCCTTACAAGCAAAGTATTACTTTGT (CRBN-P2), 5′-CCGCTCGAGTGGCCGGCGAAGGAGATCAGCAGGACG-3′ (CRBN′-P1), and 5′-CGGGATCCTCACGTTAAGCCCCAAAATTTTTGAGG-3′ (CRBN′-P2). The CRBN and CRBN′ gene consequently were cloned to pEGFP-c1 Plasmid (Clontech) at the XhoI and BamHI site. AD293 cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco) with 10% (v/v) fetal bovine serum (FBS) and 100 U/ml penicillin. The cells were incubated at 37°C in air containing 5% carbon dioxide. The generated fusion plasmids were then transfected into AD293 cells with LipofectamineTM 2000 Reagent according to the manufacturer’s protocol (INVITROGEN). The pEGFP-C1 plasmid was served as control.

Results and discussion

Sequence characterization

By large scale sequence analysis of our fetal brain cDNA library, we isolate a cDNA which is identical with the mental retardation related gene cereblon (CRBN). This cDNA was 2231 bp in length and contained an ORF of 1329 bp encoding a protein of 442 aa. The upstream in-frame stop codon suggests that this cDNA has an imtact ORF.

By comparing the CRBN cDNA sequence with the human genomic DNA, we found that the CRBN gene spanned more than 29 Kb on genomic DNA and consisted of 11 exons. We analyzed the exon-intron structure of the CRBN gene and found that all the sequences of the exon-intron junctions were consistent with the AG–GT rule (Table 1). The protein profile prediction was performed using software at the ExPASy Molecular Biology Server (http://expasy.pku.edu.cn). It suggested that CRBN had an 273-amino-acid ATP-dependent LON domain. We also found the mutation of CRBN (1,274(C > T)) interrupted an N-myristoylation site at 416–421 (GLtrSA) and eliminated two casein kinase II phosphorylation sites at 425–428 (TipD) and 429–432 (TedE). These disruptions may influence its proper subcellular targeting and alter long-term potentiation in the hippocampus.

Table 1 Nucleotide sequence of exon-intron junctions of the human CRBN gene
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The homologues of CRBN in other species were found out by using BLAST search at NCBI (http://www.ncbi.nlm.nih.gov/blast). Amino acid sequence similarity were determined with the ClustalW and the GeneDoc programs (http://www.psc.edu/biomed/genedoc Fig. 1). CRBN has a LON domain from residues 80 to 320, and this domain is conserved in all the homologs, especially at the residue position 89, 98, 99, 101, 102, 105, 106 and so on. This may suggests that these amino acid residues were very important and not changed even under the evolution pressure.

Fig. 1
figure 1

Alignment of amino acid sequences of human CRBN(NP_057386) with the homologues from other species: Xenopus tropicalis, NP_001008192; Rattus norvegicus, AAH99779; Apis mellifera, XP_395264; Tetraodon nigroviridis, CAG06776; Mus musculus, AAH69905; Macaca mulatta, XP_001100484; Tribolium castaneum, XP_970608; Canis familiaris, XP_862897; Pan troglodytes, XP_001140181; Gallus gallus, XP_414437; Drosophila melanogaster, NP_649973. Residues indicated with dark shading are identical amino acids

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Expression pattern of CRBN

By searching the Unigene database and the genome database, we found the CRBN gene matches 103 ESTs and a genomic clone (NM_016302) from chromosome 3p26.2. These ESTs are mostly from testis, lymphocyte, placenta, erythroid precursor cells, fetal brain, tumorous cells, mammary gland, embryonic stem cells, leukocyte, prostate, medulla, blood, kidney, lung and spleen.

The tissue distribution of CRBN mRNA is determined by RT-PCR. It shows an extremely high expression level in human testis; high expression in spleen, prostate, liver, pancreas, placenta and kidney; and moderate expression in lung, skeletal muscle, ovary, small intestine, peripheral blood leukocyte, colon and brain. We cannot detect the expression in heart and thymus (Fig. 2). These results are consistent with the EST distribution.

Fig. 2
figure 2

Tissue distribution of CRBN mRNA. RT-PCR analysis of human cDNA for CRBN and G3PDH (as a control). Prenormalized cDNAs from 16 human adult tissues were employed as templates in PCR reactions containing CRBN and G3PDH-specific primer as described in materials and methods

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The distribution of human CRBN was partially matched to that of rat CRBN in rat tissues [18]. Its highest expression in testis may suggest the important role of CRBN in organogenesis. Its lower expression in brain compared with rCRBN may suggest a role except for in memory and learning. rCRBN was reported to have the direct interaction with the cytosolic carboxy-terminus of the large conductance Ca2+-activated K+ (BKCa) channel α subunit as a binding protein. As expression of large conductance calcium-activated potassium channels decreases cellular protein tyrosine phosphorylation [20], CRBN might effect an important signal transduction pathways. CRBN was similar to rCRBN (amino acid identities of 93%, using BLAST2.0 at NCBI http://www.ncbi.nlm.nih.gov/blast). It was suggested that CRBN might also be involved in signal pathways, not only related to memory and long-term potentiation.

Subcellular localization of CRBN and CRBN′

By RT-PCR, we confirmed that AD293 cell line express the CRBN gene (Fig. 3 left panel). This suggests that AD293 cells could be used in CRBN subcellular localization assay. The pEGFP-C1-CRBN and pEGFP-C1-CRBN′ (1,274(C > T)) recombinant plasmids were transfected into AD293 cells and the expressed proteins were visualized under fluorescence microscope. The results demonstrate that CRBN and CRBN mutant of CRBN (1,274(C > T)) were both localized in the whole AD293 cells (Fig. 3 right panel).

Fig. 3
figure 3

CRBN obtained by RT-PCR from AD293 cells(left panel, C: CRBN). Subcellular distributions of CRBN and CRBN′ (right panel). AD293 cells transfected with pEGFP- CRBN and pEGFP-CRBN′ were stained with DAPI after fixation and observed under a fluorescence microscope (CRBN : A–C; CRBN′: D–F). pEGFP was also transfected as a control (G–I )

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It was reported that endogenous rCRBN was expressed in the juxtanuclear region and cytoplasm, but not in the nucleus [17]. Most human Lon-containing proteins are localized to the mitochondria where they selectively degrade short-lived polypeptides [15, 16]. It seems that CRBN was not only related with mitochondria [19] where CRBN participated in energy metabolism but also related with cytoplasm and nucleus. We speculate that CRBN may participate in other biological processes.

In conclusion, human CRBN cDNA was isolated. The tissue distribution of CRBN mRNA and protein subcellular localization showed that CRBN functions in several tissues and several organelles and may play some roles in many areas other than learning, memory and energy metabolism.