Department of Pathology and 2Department of Medicine, Division of Cardiology, UCLA Medical School, Los Angeles, CA, USA, and 3Department of Biochemistry and Molecular Biology and Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602
Received on June 27, 2000; revised on September 8, 2000; accepted on September 12, 2000.
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Abstract |
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Key words: lectin/endothelial cells
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Introduction |
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We isolated the cDNA encoding XL35 from a Xenopus oocyte cDNA library (Lee et al., 1997) and determined that the amino acid sequence it encodes did not display the C-type lectin motif, although it does require calcium for binding (Drickamer, 1993
). We have recently identified sequence homologs of XL35 and describe here the characterization of two cDNAs, each with an unusual tissue-specific expression pattern, termed HL-1 and HL-2. One of the homologs, HL-1, is expressed exclusively in the vascular endothelial cells in a unique set of tissues, as well as in the endocardium. The other, HL-2, is expressed only in small intestine. These and other results demonstrate the existence of a family of proteins with homology to XL35.
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Results |
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Chromosomal localization
To analyze the chromosomal localization of the HL-1 and HL-2 genes, FISH (fluorescence in situ hybridization) analysis was performed using genomic clones encoding HL-1 and HL-2 isolated by Genomic Systems, Inc. Both clones hybridized to chromosome 1q band 23 (data not shown). Recently, fragments of genomic sequence in the human genome database confirmed our results that localized both HL-1 and -2 at chromosome 1q 2223.5. One cosmid clone of 190 kb contained both HL-1 and -2 sequences. This result suggests that the two sequences arose most likely from a gene duplication event that ultimately resulted in proteins with similar sequences, but with tissue-specific promoters. Intriguingly, the genes that encode the selectin family of lectins are found between 1q22 and 1q25 (Watson et al., 1990).
Tissue distribution of HL-1 and HL-2 mRNA
The tissue transcript levels of HL-1 and HL-2 were determined by Northern blot analysis using the 161 bp radiolabeled probe described above. The major transcripts of both HL-1 and HL-2 were 1.3 kb in length (Figure 3A,B). The transcript of HL-1 was observed in relatively very high levels in heart, colon, small intestine, and thymus, with lower levels in ovary, testis, and spleen (Figure 3B). Additional Northern analyses also showed strong signals in lymph node and stomach (data not shown). By stark contrast to HL-1, the transcript of HL-2 was detectable exclusively in small intestine (Figure 3A). The size of this transcript was 1.3 kb, similar to that of HL-1. HL-2 is most likely more abundant than HL-1 in small intestine, since 6 of 7 clones isolated from the small intestine cDNA library contained HL-2 cDNA sequences.
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Cell type specific expression of HL-1
To determine which cell types expressed HL-1, immunohistochemistry was performed on human tissue sections using rabbit polyclonal antisera prepared against affinity-purified XL35. Striking results were obtained from colon, liver and thymus sections using anti-XL35 primary antisera and peroxidase-conjugated secondary antibody. The antisera specifically and intensively stained endothelial cells lining the blood vessels in these tissues (Figure 4), while pre-immune serum staining was negative. In all tissues, the labeling appeared throughout the endothelial cells, not just adsorbed to the cell surface. In the colon, endothelial cells lining vessels in both the submucosa (arrows) and the lamina propria (arrowheads) were stained with high intensity (Figure 4B,C), consistent with the high level of transcripts observed by Northern analysis (Figure 3A). Staining of liver revealed intense immunoreactivity in both the small hepatic arteries and hepatic veins in the portal tract (Figure 4E, arrows), as well as weaker reactivity of endothelial cells lining the hepatic sunusoids (Figure 4E, arrowhead). In thymus, prominent staining of vessels was noted (Figure 4G). An identical pattern of staining was noted in tonsil, a specialized lymph node, including high endothelial venule cells (data not shown). In heart sections, the endocardium, derived from endothelium, and heart blood vessel endothelial cells showed immunoreactivity, with weak interstitial staining of myocardium (data not shown). These results show that the HL-1 protein is expressed in the endothelial cells in specific tissues, consistent with the tissue specific mRNA expression pattern we observed.
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Discussion |
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It was quite interesting, therefore, to find homologs of XL35 in adult human tissues. The amino acid sequences of the lectins predict signal sequences for secretion, and XL35 is known to be secreted at fertilization. If the putative signal sequences, which are very divergent, are excluded from consideration, the identity between the HL-1 and XL35 amino acid sequences is 63% and the similarity is 75%. Moreover, eight conserved cysteine residues are observed between the two sequences. HL-2 shows slightly less identity and similarity to XL35 than does HL-1. Although we have no direct evidence as yet, these structural considerations suggest HL-1 and HL-2 may indeed function as lectins. The oligosaccharide binding specificity of XL35 is known in general terms; however, the location of the amino acids that constitute its binding site is as yet unknown. Western blots of partly purified rat heart homogenate using polyclonal antisera against XL35 identify several protein bands with molecular weights roughly similar to those of HL-1 in human heart aortic endothelial cells (Figure 5). To determine if the homolog in rat heart has a ligand binding specificity similar to that of XL35, the rat heart extract was applied in the presence of Ca2+ to an affinity column of immobilized melibiose, an -linked disaccharide to which XL35 binds (Roberson and Barondes, 1982
). Bound proteins were eluted with EDTA, and the eluted proteins were analyzed by Western blotting using anti-XL35 antisera (Lee, 1997
). No lectin-related proteins bound to the melibiose column, showing that the rat heart homolog most likely does not bind the same disaccharide as does XL35. Since only very minor differences in the amino acid sequence of lectins can yield proteins that differ in their binding specificity, no prediction can be made whether HL-1 and HL-2 bind the same ligand (Drickamer, 1992
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No C-type lectin motif is found in XL35 (Drickamer, 1993), although it has calcium-dependent oligosaccharide ligand binding activity. Likewise, HL-1 and -2 sequences do not contain this motif. A small region of XL35 and the human homologs, however, does contain a fibrinogen-like motif, Figure 7. Members of the Ficolin/Opsonin/p35 lectin family contain significant homology to fibrinogen, in addition to collagen-like domains (Kilpatrick et al., 1997
; Ohashi and Erickson, 1997
). Proteins in this family are found in serum, some have been shown to have lectin-like activity, and they have been postulated to function generally in pathogen opsinization (Matsushita et al., 1996
). It has been hypothesized that the carbohydrate-binding domain present in this family of lectins is contained at least partially in the small domains they share with fibrinogen. As depicted in Figure 7, HL-1, HL-2, and XL35 all contain a portion of a fibrinogen-like motif found in the Ficolin/Opsonin/p35 family, suggesting that this region may encode their carbohydrate binding domains. Both ficolin and the Hakata antigen, a circulating serum lectin that is a member of the Ficolin/Opsonin/ p35 family (Sugimoto et al., 1998
), however, also contain this small fibrinogen-like motif shared with HL-1, HL-2, and XL35. Neither of these proteins, however, require calcium for their carbohydrate binding activity. It is unlikely, therefore, that this domain found in XL35 is predominantly responsible for its ligand binding activity, but further experiments are obviously necessary to identify its oligosaccharide binding site and the putative binding sites in HL-1 and -2.
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The presence of HL-1 in vascular endothelial cells, coupled with the detection of the HL-2 transcript in small intestine and a transcript similar to the human lectins in mouse small intestine, demonstrates defined, unique expression patterns for these homologs. Members of this family with sequences similar to the Xenopus cortical granule lectin clearly must have functions distinct from that of the cortical granule lectin. Experiments to define these functions are in progress.
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Materials and methods |
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Detection of partial cDNA sequences of human homologs
XL-35 cDNA and peptide sequences were used to search protein and DNA sequence databases, and a single entry was identified from non-Xenopus sources. The sole sequence match was a fragmentary 251 bp cDNA sequence obtained from a human heart cDNA library (GenBank, Accession number Z36760) as an expressed sequence tag (EST) (Figure 1). Using the human EST sequence, we designed oligonucleotide primers (sense primer: 5'-CAGACCTTCTGTGACATGACCTCT-3' and antisense primer: 5'-AAGATGCCCAGGTCCTTGGCCTGG-3'). The human placenta, spleen, and liver cDNAs were subjected directly to PCR reactions in a 25 µl reaction volume containing 1 µM of each primers, 10 ng of each cDNA, 0.2 mM of each dNTP, 10 mM TrisHCl, 50 mM KCl, 1.5 mM MgCl2, and 1.5 units of Taq polymerase. The thermal cycling conditions were 92°C, 1 min; 55°C, 1 min; and 72°C, 1 min, for a total of 35 cycles. The amplification products were separated on a 0.7% agarose gel and purified using Sephaglas DNA purification kit (Pharmacia). Each amplimer was subcloned into the pCR II vector using the TA cloning kit (Invitrogen) and fully sequenced. The deduced peptide sequences from the PCR products, the human heart EST sequences, and the XL-35 cDNA clone were aligned to compare their sequence identities. The cDNA amplimers from the human tissues were essentially identical to the human heart EST sequence.
cDNA library screening
A human small intestine 5'-stretch gt10 cDNA library was purchased from Clontech and was screened by plaque hybridization using the 251 bp amplification product of the human liver cDNA as a radiolabeled probe. The cDNA amplimer was isolated on a 1% agarose gel, purified, and labeled using [
-32P]-dCTP and the Mega-Prime labeling kit (Amersham). Plaque lifts were screened by standard formamide/SSC hybridization conditions using Hybond-N filters (Amersham) (Sambrook et al., 1989
). Positive phage clones identified by plaque hybridization were purified through four rounds of plaque purification. Lambda DNA from positive clones was isolated from 100 ml cultures by polyethylene glycol precipitation followed by chromatography over a Qiagen Tip-100 column as described by the manufacturer (Qiagen). The full-length inserts from the clones were isolated by PCR amplification using primers specific for the flanking vector sequences. A total of 13 clones were identified with insert sizes varying from 0.7 to 2.4 kb. Seven cDNA inserts >1.2 kb were subcloned into the pCR II vector and fully sequenced. Comparison of the sequences of the clones indicated that six of the clones were identical (named HL-2) and one clone (HL-1) had a different sequence (Figure 1).
DNA sequence analysis
All cDNA clones and genomic clones were sequenced by using Taq polymerase in the dideoxy dye-terminator reaction using T7 or SP6 polymerase primers, or synthetic primers. The sequencing reactions were analyzed on an Applied Biosystem 373A DNA sequencer (Molecular Genetics Instrumentation Facility, University of Georgia). DNA sequence data were assembled into a contiguous sequence database by the method of Staden (1987)). A sequence similarity search between protein or DNA sequences was performed using the Bestfit, Pileup, Fasta, and Tblast programs of the University of Wisconsin Genetics Computer Group (GCG software, version 8.0).
Isolation of genomic DNA clones
An arrayed human genomic library in the P1 vector (Genome Systems, Inc., St Louis, MO) was screened by a PCR approach (Pierce et al., 1992; Shepherd et al., 1994
) using primer pairs specific for the respective genes. Pools of clones were screened by PCR amplification using primers for HL-1 (sense primer: 5'-ATACTTTCCAGAGGCCAGTCCCCAG-3', antisense primer: 5'-AGGTCTGGGTTCCCTCCCACAAAAC-3') or HL-2 (sense primer: 5'-GTTCTTCCCACAGGGCAAACCCCGT-3', antisense primer: 5'-TCTGCCCTGACACCGGAGAGCTCTG3'). The PCR reactions were performed in a 25 µl reaction volume containing 1 µM of each primers, 50 ng of human genomic DNA, 0.2 mM each dNTP, 10 mM TrisHCl, 50 mM KCl, 1.5 mM MgCl2, and 1.5 units of Taq polymerase. The thermal cycling conditions were: 94°C, 1 min; 65°C, 1 min; and 72°C, 1 min, for a total of 35 cycles. An amplimer of
160 bp was obtained from PCR reactions from the respective template (dotted-line sequences in (Figure 1). The sequence of each amplimer was identical to that of each cDNA clone, which implied that the region of amplification in the lectin genes did not contain introns. The same primers and PCR reaction conditions were used for the screening of genomic DNA clones. Two genomic clones were isolated for both HL-1 and HL-2 and each was partially sequenced using synthetic primers designed based in the sequence of the cDNA clones. Sequence data and results of PCR amplification indicated that each clone contained either HL-1 or HL-2, but not both genes.
Northern blot analysis
The cDNA-specific PCR products for HL-1 or HL-2 obtained from PCR of human liver cDNA were labeled using [-32P] dCTP and the Mega-Prime labeling kit (Amersham) according to the manufacturers instructions. The 161 bp amplimers probes for HL-1 and HL-2 obtained from PCR were only 63.9% identical in sequence (58/161 mismatches). The radiolabeled cDNA fragments were used to probe Northern blots of various human tissue poly(A+) RNAs (human multiple tissue Northern blot, Clonetech). The blots were probed using a prehybridization buffer consisting of 0.5 M Na phosphate, pH 7.0, 1 mM EDTA, 7% SDS, 10 mg/ml of fatty acid free BSA, and 100 µg/ml denatured herring sperm DNA. The hybridization was using the identical buffer, except for the addition of denatured radiolabeled probe. Prehybridization was performed for 2 h at 65°C, and hybridization was performed overnight at 65°C. The blots were washed three times at 65°C for 15 min in 50 ml of 40 mM Na phosphate, pH 7.0, 1% SDS, 1 mM EDTA, and 0.5% fraction V BSA and an additional three times at 65°C for 15 min in the same buffer excluding BSA. The blots were then washed in 50 ml of 1 M phosphate buffer, pH 7.0, at room temperature for 10 min and data collected using a Molecular Dynamics phosphoimager or autoradiography.
Immunoblot analysis
Polyclonal rabbit antisera against affinity-purified XL35 was prepared similarly to that described by Barondess laboratory (Roberson and Barondes, 1983). Antisera was specific for XL35 on Western blots of oocytes, and reacted with N-glycanase-treated XL35. Human thymic stroma, obtained after removal of thymocytes, was extracted by homogenization in lysis buffer (10 mM Tris, pH 7.3, 130 mM NaCl, 5 mM CaCl2, 1 mM PMSF, 0.5% NP-40). Cultured human aortic endothelial cells (HAEC), either stimulated with LPS (Baum et al., 1995b
) or mock stimulated controls, were lysed by brief vortexing in lysis buffer. Nuclei were removed by centrifugation for 2 min at top speed in a microcentrifuge. Two hundred micrograms of protein were loaded per lane, and electrophoresed under reducing conditions. Immunoblot analysis was performed as described previously (Baum et al., 1995b
). The blot was probed with rabbit polyclonal antiserum against native XL35 diluted 1:1000 in 100 mM Tris, 150 mM NaCl, 0.1% Tween (TBST) with 1% nonfat dry milk. After repeated washes with TBST, the blot was incubated with horseradish peroxidase conjugated goat anti-rabbit IgG (Bio-Rad), and visualized by chemiluminescence (ECL, Amersham).
RT-PCR analysis
Total RNA was purified from the indicated cell types using Trizol reagent (Gibco BRL), according to the manufacturers directions. Two micrograms of total RNA was reverse transcribed (Ready to go T primed first strand kit, Pharmacia). Two microliters of the first strand reaction was used as template for PCR amplification. PCR was performed using 200 µM dNTP, 1 µM each oligonucleotide primer, and Taq polymerase and buffer (Stratagene). The sequence of the 5' primer was 5'CAGACTACCCAGAGGGGGACGGCAACTGG3'. The sequence of the 3' primer was 5'CTCCACCAATGCAGTGGTGCTCAGTGTTAC3'. The thermal cycling conditions were: 94°C x 45 s, followed by 72°C x 2 min for a total of 40 cycles followed by a final extension for 10 min at 72°C.
Immunohistochemical analysis
Experiments were performed on 6 mm sections of the indicated tissues as previously described (Baum et al., 1995a), using rabbit polyclonal antiserum against native XL35 diluted 1:1000, and the sections were counter-stained with hemotoxylin. Control sections were incubated with a similar dilution of preimmune serum.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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