CL-46, a Novel Collectin Highly Expressed in Bovine Thymus and Liver

cDNA cloning

RNA was isolated from 250 mg of bovine thymus using the TRI-reagent kit according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO). cDNA was synthesized from 3 μg of total RNA with Superscript II H⁻ reverse transcriptase (Life Technologies, Grand Island, NY) according to the recommended conditions and with the specific primer 5′-CCATAGGAGGCCTGGCTT-3′.

Two microliters of the 20-μl cDNA reaction was used as template in the following PCR to amplify a full-length CL-46 transcript. In a total volume of 30 μl, a PCR was performed using the primers 5′-GCACTTCAGACTCCAGTACTAGCCTGTG-3′ and 5′-CCATAGGAGGCCTGGCTT-3′ and using Pwo polymerase (Roche, Mannheim, Germany) according to the conditions recommended by the manufacturer. After 2 min initial denaturation at 94°C, 0.5 U of Pwo polymerase was added and 10 cycles of 94°C for 45 s, 69–60°C for 30 s, 72°C for 3 min were performed with a decreasing annealing temperature of 1°C per cycle. Twenty-five cycles at 94°C for 45 s, 59°C for 30 s, 72°C for 3 min followed. Half of a unit of Taq polymerase (Life Technologies) and fresh dNTPs were added to a final concentration of 1 mM and were followed by a final extension of 6 min at 72°C. The product was analyzed by agarose gel electrophoresis and purified from the excised gel plug with Qiaex II gel extraction kit (Qiagen, Hilden, Germany). Ten nanograms of the product was ligated into the PCRII-vector and heat shock transformed into INVaF′ Escherichia coli using the Original TA cloning kit (Invitrogen, Groningen, The Netherlands). Plasmids were purified using the Wizard plus SV Miniprep purification system (Promega, Madison, WI) and sequenced.

RT-PCR

RNA was isolated from various tissues and cDNA synthesis was conducted as described above using the primers 5′-TGTCATTCCACTTGCCCTCCC-3′ and 5′-TTCTCTTCCATAAACATTCTCA-3′ specific for CL-46 and bovine β-actin, respectively. Between 1 and 3 μg of RNA was used as template in the synthesis of cDNA. A volume of 1–3 μl of the first-strand synthesis was used as template in the following PCRs. β-Actin was amplified by a PCR using the primers 5′-CTGGCCGGGACCTGACA-3′ and 5′-CACGCCGCAGTCCATTTAG-3′ according to standard conditions for the Taq polymerase (Life Technologies). After 2 min of initial denaturation, 0.2 U of Taq was added to a 30-μl reaction. Twenty-five cycles of 94°C at 45 s, 54°C for 30 s, 72°C for 45 s, and a final extension at 72°C for 1.5 min followed. CL-46 was amplified as described above using the primers 5′-CTCAAGCAGCGGGTGACC-3′ and 5′-CTCTGGTTGTCCAGCATTGT-3′. Amplification comprised initial denaturation at 94°C for 2 min, addition of 0.2 U of polymerase, and 10 cycles at 94°C for 45 s, 61–52°C for 30 s, 72°C for 30 s, with a 1°C decreasing annealing temperature per cycle. Nineteen or 27 cycles at 94°C for 45 s, 51°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 1.5 min followed. The products were analyzed by agarose gel electrophoresis.

Genomic cloning

A CL-43-derived probe, spanning nucleotide 633-1026 of the CL-43 cDNA sequence (GenBank accession number X75912), was obtained by PCR amplification with the primers 5′-GGCCTCCCCACGCTCTTCA-3′ and 5′-CCTTCTGGCCTCATCCTGTGG-3′ and a previously isolated CL-43 cDNA clone as template (19). The PCR consisted of 4 min of initial denaturation at 94°C followed by 30 cycles of 94°C for 45 s, 56°C for 30 s, 72°C for 30 s, and a final extension step at 72°C for 1 min. The product was purified using the Qiaex II gel extraction kit and labeled with [³²P]dCTP using an oligolabeling kit (Amersham Pharmacia Biotech, Piscataway, NJ) and random primers according to the recommended procedure. Approximately 2 × 10⁵ plaques of a bovine genomic EMBL3 lambda phage library (Clonetech, Palo Alto, CA) were screened with the [³²P]-labeled probe. The final high-stringency wash was done in 0.3× SSC at 55°C for 20 min. Positive clones were replated and rescreened twice to uniformity. Phage DNA was isolated, digested with SacI, and analyzed by agarose gel electrophoresis. Fragments not observed in a similar digestion of empty EMBL3 vector were purified using the QIAX II gel extraction kit. Purified fragments (20–100 ng) were ligated into pBluescript SK+ vector (25 ng), previously digested with SacI and treated with calf intestinal phosphatase, and heat shock transformed into chemically competent XL-10 E. coli. Plasmids were purified and sequenced as above. To assemble the nonoverlapping sequences of the subclones, three PCRs overlapping the junctions were performed with the following primers: 5′-CGGACCAAAGGGAGACACTG-3′(P1), 5′-GTCCCTGCTGCTCACACCAC-3′(P1), 5′-AGAGGAAAAGGGCAGTGGGTCAG-3′(P2), 5′-GGGTGAGGAGAAAGGGGAGGAC-3′(P2), 5′-GAGCATGAATGACATCTCCAC-3′(P3), and 5′-AGAAGAGAAAAGGGAAGGATGT-3′(P3). Fifty nanograms of purified phage DNA was used as template and denatured at 94°C for 4 min. The amplification comprised 30 cycles of 94°C for 45 s, 54°C(P1)/59°C(P2)/49°C(P3) for 30 s, 72°C for 1 min (P1) or 30 s (P2, P3) and was followed by a final extension step at 72°C for 2 min. Products were analyzed by agarose gel electrophoresis, purified, and sequenced. The 5′-upstream sequence was analyzed for potential cis-regulatory elements according to Quandt et al. (20) and using matrices from the Transfac 4.0 database (Biological Databases, Braunschweig, Germany). The analysis was high stringent requiring a complete core-match and a relative fit of >0.9.

Chromosome localization

Genomic DNA (20 ng) of 90 hamster/cow somatic cell hybrids (21) was used as template in PCRs with the primers 5′-GCAGGTGCTGTAAAATCATATTC-3′ and 5′-GGGATGTTGGCAGCTCA-3′. Amplification comprised a 4-min initial denaturation, 10 cycles at 94°C for 45 s, 61–52°C for 30 s, 72°C for 25 s, with a decreasing annealing temperature of 1°C per cycle, and followed by another amplification of 25 cycles at 94°C for 45 s, 51°C for 30 s, 72°C for 25 s, and a final extension at 72°C for 2 min. Products were analyzed using agarose gel electrophoresis.

Expression of rCL-46 in Pichia pastoris

cDNA corresponding to the α-helical coiled-coil neck region and the CRD of CL-46 was amplified by a PCR using the primers 5′-TACGTAGTCAATGCTCTCAAGCAGCGG-3′ and 5′-CCTAGGTCAAAACTCGCAGATCACAAGGAG-3′ that insert SnaBI and AvrII restriction sites in the 5′- and 3′-end, respectively. Ten nanograms of pCRII plasmid encoding full-length CL-46 cDNA was used as template in a 60-μl PCR with 0.5 U Pwo polymerase. Amplification comprised 2 min of initial denaturation at 94°C, 30 cycles consisting of 94°C for 45 s, 57°C for 30, 72°C for 1 min, and a final extension at 72°C for 2 min. The product was isolated using the Qiaex II gel extraction kit as before and ∼300 ng of DNA was digested with SnaBI and AvrII. After agarose gel electrophoresis, the product was purified using the Qiaex II gel extraction kit, and 25 ng was ligated into 100 ng of pPIC9K vector (Invitrogen), previously digested with SnaBI and AvrII and treated with calf intestinal phosphatase. Ligated plasmids were heat shock transformed into XL-10 E. coli as described above and purified from a sequence-verified clone. Eight micrograms of SacI-linearized plasmid was transformed into P. pastoris (GS115) by electroporation (22). Clones were double selected by growth on histidine-deficient plates and plates with increasing concentrations of geneticin according to the manufacturer’s protocol. Expression was done in medium made of 200 mM phosphate buffer, 1.34% yeast nitrogen base (QBIOgene, Middlesex, U.K.), 0.5% methanol, and 4 × 10⁻⁵% d-biotin (pH 6) and spanned 4 days.

Purification of rCL-46

EDTA was added to 1 liter of P. pastoris culture to a final concentration of 3 mM, and the pH was adjusted to 5. The culture was centrifuged at 8000 × g for 10 min at 4°C and the supernatant was filtered through 0.45-μM filters. It was loaded onto an SP-Sepharose column (20 ml; Amersham Pharmacia Biotech). The column was washed with 200 ml of buffer made of 30 mM MES, 0.5 mM EDTA, 0.01% Tween 20 (Tw), pH 5 and eluted with a similar buffer with 800 mM NaCl. Tris and CaCl₂ were added to final concentrations of 20 and 4 mM, respectively, and the pH was adjusted to 7.4. The eluate was loaded onto a 20-ml column made of TSK HW/75 (F) (Tosoh, Tokyo, Japan) derivatized with N-acetyl-d-glucose-amine (GlcNAc), using divinyl-sulfone as previously described (23). After washing with 200 ml of TBS/Tw/Ca²⁺ (10 mM Tris, 150 mM NaCl, 1 mM CaCl₂, 0.01% Tw, pH 7.4) the column was eluted with TBS/Tw/EDTA (TBS/Tw/Ca²⁺ with 2 mM EDTA instead of CaCl₂).

Preparation of Abs

Rabbits were immunized s.c. with 25 μg of purified rCL-46 in Freund’s complete adjuvant. The following monthly boosts were done with the same Ag amount in Freund’s incomplete adjuvant and antisera were collected 2 wk after a boost. Abs were purified using a 1-ml Protein G fast flow column (Amersham Pharmacia Biotech).

Carbohydrate specificity

Microtiter wells (Maxisorb; Nalge-Nunc, Roskilde, Denmark) were coated overnight at room temperature with 200 ng of GlcNAc-BSA (Sigma-Aldrich) in 100 μl of buffer made of 15 mM Na₂CO₃ and 35 mM NaHCO₃. Plates were emptied and incubated for 2 h at room temperature in TBS/Tw/Ca²⁺ with 0.1% (w/v) BSA and washed in TBS/Tw/Ca²⁺. Dilutions of monosaccharides in 50 μl of TBS/Tw/Ca²⁺ were added in duplicate to the wells. Negative and positive controls consisting of TBS/Tw/EDTA or TBS/Tw/Ca²⁺ without monosaccharides were included. Purified rCL-46 at 4 μg/ml TBS/Tw/Ca²⁺ was then added in duplicate 50-μl volumes. The solutions were mixed on a shaking platform and incubated overnight at 4°C. The saccharides tested comprised mannose (Man), α-methyl-d-Man (αMeMan), d-mannosamine (ManN), N-acetyl-d-mannosamine, (ManNAc) d-glucose, (Glc), α-methyl-d-glucose (αMeGlc), d-glucosamine (GlcN), GlcNAc, d-galactose (Gal), d-galactosamine (GalN), N-acetyl-d-galactosamine (GalNAc), d-fucose (Fuc), l-fucose (l-Fuc) and maltose (Mal). All were tested at concentrations ranging from 0.1 mM to 100 mM. The wells were washed in TBS/Tw/Ca²⁺ and incubated at room temperature for 3 h with rabbit-anti-rCL-46 IgG diluted to 2 μg/ml TBS/Tw/Ca²⁺. Wells were washed in TBS/Tw/Ca²⁺ and incubated with alkaline phosphatase-conjugated goat anti-rabbit Ig (DAKO, Glostrup, Denmark) diluted to 0.25 μg/ml TBS/Tw/Ca²⁺. After incubation at room temperature for 3 h, wells were washed with TBS/Tw/Ca²⁺ and developed with para-nitro-phenyl phosphate. The background was defined as the average signal of three wells incubated with rCL-46 in the presence of EDTA; the maximum signal was that obtained in buffer without monosaccharide. Each saccharide was tested in three different experiments, and various combinations of five different monosaccharides were tested on the same microtiter plate to determine their individual ranking.

SDS-PAGE

SDS-PAGE was done in a discontinuous buffer system on 4–20% gradient gels (24). Proteins were stained with Coomasie brilliant blue R250 and molecular mass was estimated by comparison with prestained marker proteins (Mark 12 from Invitrogen).

DNA sequencing

PCR products or plasmid preparations were sequenced with the Prism Ready Reaction BigDyeDeoxy Terminator sequencing kit (PE Applied Biosystems, Alleroed, Denmark) using the recommended conditions. Samples were subjected to electrophoresis on an ABI prism 310 Genetic Analyzer, and data was analyzed with the ABI Prism Software version 2.1.1 (PE Applied Biosystems).

N-terminal sequencing

Recombinant CL-46 was subjected to SDS-PAGE and electrophoretic transfer at 7.5 V/cm for 10 h to Problot membranes (PE Applied Biosystems) in buffer made of 10 mM 3-cyclohexylamino-1-propanesulfonic acid, 10% (v/v) methanol at pH 11. The protein band was visualized with Ponceau S, excised, and sequenced on a PE Applied Biosystems 470/120A sequencer.

Identification and cloning of a novel bovine collectin (CL-46)

During the screening of a genomic phage library with a probe encoding the CRD of CL-43, we isolated three clones that encoded a putative novel collectin gene with high sequential similarity to bovine SP-D, conglutinin, and CL-43. Sequences obtained from the clones made it possible to establish a specific PCR, which we used in RT-PCR. We found that the new gene was transcribed in the thymus, and using a new pair of primers we amplified the entire open reading frame from thymus RNA. This open reading frame codes for a polypeptide of 371 aa residues. The total cDNA comprised 65 nucleotides of 5′-untranslated sequence, 1116 nucleotides encoding the protein with intact stop and start codons, followed by a 75-nucleotide 3′-untranslated sequence with a complete polyadenylation site (Fig. 1⇓). The deduced amino acid sequence of the cDNA (Fig. 1⇓) revealed a collectin structure composed of a signal peptide of 20 aa residues, an N-terminal segment of 25 aa residues, 57 Gly-Xaa-Yaa repeats, a neck region of 28 aa residues, and a CRD of 127 aa residues. Assignment of the cleavage site of the signal peptide was done by sequence comparison to bovine SP-D and conglutinin. Two cysteine residues were found in the N-terminal segment and a potential N-glycosylation site was found at aspargine 90 in the collagen-like region. Further alignment of the amino acid sequence with the sequence of other collectins showed that the CRD of CL-46 posses all necessary and conserved residues involved in calcium and carbohydrate binding (Fig. 2⇓). The cDNA sequence of CL-46 has been submitted to GenBank (accession number AF509590).

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FIGURE 1.

Nucleotide and deduced amino acid sequences of CL-46 cDNA. Nucleotides and amino acid residues are numbered in 5′ to 3′ direction and N-terminal to C-terminal direction, respectively. The translation start codon (ATG) and the polyadenylation signal (AATAAA) are underlined.

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FIGURE 2.

Alignment of collectin carbohydrate recognition domains. The CRDs of human CL-L1, human SP-A, human MBL, human SP-D, porcine SP-D, bovine SP-D, CL-46, conglutinin, and CL-43 were aligned. Black-shaded residues are identical and dashes indicate gaps included to improve the alignment. The 18 conserved residues found in all collectins are indicated with arrows.

RT-PCR analysis of CL-46 expression in various tissues

To examine the expression of CL-46 we used RT-PCRs on RNA isolated from 20 different tissues obtained from a 2.5-year-old cow immediately after slaughtering (Fig. 3⇓). We normalized the amount of RNA with the expression of β-actin. Amplified products of β-actin were sequenced and identical with the partial sequences of bovine β-actin (GenBank accession numbers K00622 and K00623). To exclude contamination with genomic DNA, the CL-46-specific RT-PCR amplified a product spanning exons 7 and 8 (see Genomic characterization). After a touchdown amplification and 19 cycles of PCR, we observed expression of CL-46 mRNA in the thymus and the liver. Another eight rounds of amplification showed additional expression in the mammary gland, the rumen, the reticulum, the omasum, the abomasum, the small intestine, and the large intestine. Sequencing the amplified products verified that they derived from CL-46. Unlike what we observed with many other primers, the chosen primer pair amplified only CL-46 and not transcripts of SP-D, conglutinin, and CL-43.

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FIGURE 3.

RT-PCR analysis of CL-46 expression. CL-46 transcripts were amplified by RT-PCR using total RNA isolated from bovine tissues. Bovine β-actin was amplified from the same tissues and used as a standard to correlate the amount of RNA.

Genomic characterization

Using a probe corresponding to the neck and CRD of CL-43 (see Materials and Methods), we isolated five EMBL3 lambda phage clones. Sequencing a product amplified by PCR showed that one clone contained the CL-43 gene. Restriction digestion and subcloning of the others showed that three contained the CL-46 gene and one contained a collectin-like pseudo-gene with a disrupted reading frame in the exon that otherwise encodes the CRD (data not shown). After restriction analysis, we selected one clone for further analysis. Assembling the sequences of the derived SacI subclones and the overlapping PCR products (Fig. 4⇓B) showed that the clone contained the complete CL-46 gene. We characterized ∼14 kb of the insert in the phage clone and observed that the exons encoding CL-46 mRNA are contained within a genomic sequence comprising 9799 nucleotides. The first translated exon, exon 2, encodes the signal peptide, N-terminal segment, and the first seven Gly-Xaa-Yaa repeats of the collagen-like region. The remaining collagen repeats are encoded by exons 3–6. Exons 7 and 8 encode the α-helical neck region and the CRD, respectively (Fig. 4⇓C). There was 100% identity between the sequence of our cDNA clone and the exonic sequences contained in the genomic clone. All intron boundaries obliged the GT-AG rule defining donor and acceptor sites for splicing out introns (Fig. 4⇓D). In addition to the transcribed sequence, we characterized 966 nucleotides of the potential promoter region located 5′-upstream of the first exon and 3252 nucleotides located downstream of the 3′-untranslated sequence. Three potential TATA boxes and two corresponding CAAT boxes were found in the beginning of the promoter region. The presence of multiple TATA boxes indicates that transcription may start at different locations. However, we were not able to amplify any products by RT-PCR using RNA isolated from the thymus and different forward primers located between the first TATA box (−4) and second or third (−859 or −870). Several cis-regulatory elements could be assigned to the sequence using homology search to known matrices (Table I⇓) (20). Potential AP-1 and NF-1 sequences were found in proximity of the TATA boxes and CAAT boxes (25). Many potential binding sites for the transcription factors Ikaros2 and the nuclear factors in activated T cells (NF-ATs) were found (26, 27). We also identified potential sites for NF-κB, c-Rel, and the gut-enriched Krueppel-like factor (28, 29). Besides the sites listed in Table I⇓, several potential sites for the factors, Gata1-3, Lmo2, MZF1, and S8, were also identified (30, 31, 32, 33). In the characterized 3′-downstream region we found no open reading frames of known origin. At ∼1.6 kb 3′-downstream, we found a transposon sequence with high homology to the Tn10 IS10 left transpotase of Shigella flexneri (GenBank accession number AF162223). Although it also showed homology with transposons found in other species, we encountered no complete match to any bovine transposons. Sequences of the whole gene including introns have been submitted to GenBank (accession number AF509589).

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FIGURE 4.

The CL-46 gene. A, Chromosomal localization to BTA 28 at position q1.8. B, Strategy of cloning. Subcloned fragments (S1–S5) and amplified PCR products overlapping the junctions (P1–P3) are shown. Numbers are relative to the first transcribed nucleotide in exon 1. C, Genomic organization. Exons are designated U (5′-untranslated sequence (exon 1)), SNC (signal peptide, N-terminal segment and collagen-like region (exon 2)), C (collagen-like region (exons 3–6)), α (α helical neck region (exon 7)), and CRD/U (CRD and 3′-untranslated sequence (exon 8)). The sizes of exons are indicated with numbers (bp). D, Exon and intron boundaries. Potential TATA boxes and CAAT boxes in exon 1 are underlined with straight or dashed lines, respectively. The methionine start codon in exon 2 and the polyadenylation signal in exon 8 are underlined. Capital letters symbolize transcribed sequence and numbers relate to the beginning of transcription. Complete sequence of both exons and introns (data not shown) has been submitted to GenBank (accession number AF509589).

Chromosomal localization of CL-46

Screening DNA isolated from hamster/cow somatic hybrids using PCR revealed that 22 hybrids of 90 contained the gene. No amplification of products was seen in DNA isolated from Chinese hamster ovary cells. We considered the applied PCR to be specific for CL-46 because the sequence of products corresponded to CL-46 and showed no contamination of other products. Based on previous characterizations of the hybrid panel, the CL-46 gene could be linked with a log₁₀ of odds score of 7 to the microsatellite marker ILST099, which is located on chromosome 28 at the distal position q1.8 (Fig. 4⇑A).

Recombinant expression of rCL-46

The α-helical coil-coiled neck region, starting from valine 218 (Fig. 1⇑), and CRD of CL-46 were expressed in P. pastoris (GS115). A clone, capable of growth on histidine-deficient plates and on plates with 1.5 mg of geneticin per ml of agar, was chosen for expression. N-terminal analysis showed cleavage of the α-secretion signal at the expected site, leaving the P. pastoris-derived sequence Glu-Asp-Glu-Asp-Tyr at the N terminus of the rCL-46. We purified 1–2 μg of rCL-46 per ml of culture, but the concentration in the medium was at least 10-fold higher, probably due to the presence of incorrectly folded rCL-46 that did not bind to the GlcNAc-Sepharose column. Purified rCL-46 showed a significant increased mobility in SDS-PAGE, corresponding to an increase in apparent molecular mass from 17 to 20 kDa when it was reduced (Fig. 5⇓A). The shift and the uniformity of it indicate that purified rCL-46 contains the intradisulfide bridges necessary for correct folding.

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FIGURE 5.

rCL-46 and carbohydrate specificity. A, SDS-PAGE of purified rCL-46 under nonreducing conditions (N) and reducing conditions (R). B, Carbohydrate inhibition of CL-46 binding to GlcNAc-BSA. The I₅₀ values refer to the concentration of carbohydrate required for 50% inhibition (mM). Values in parenthesis are calculated relative to N-acetyl-glucoseamine.

Carbohydrate selectivity

The potencies of various saccharides to inhibit the binding of purified rCL-46 to coated GlcNAc-BSA are given in Fig. 5⇑B. Concentrations of saccharide resulting in 50% inhibition are the average from three experiments and had variations of <6%. Binding to GlcNAc-BSA was stable in the presence of 1 M NaCl and at pH 6.0–9.0. Calcium could not be substituted with Cu²⁺, Mg²⁺, Mn²⁺, or Zn²⁺ (data not shown). The selectivity of CL-46 is compared with the binding selectivity of other collectins in Table II⇓.