(Received for publication, June 23, 1995; and in revised form, October 5, 1995)
From the
It is unclear whether the six known human defensin peptides are all encoded by separate genes or whether some of them are allelic. Three of the peptides, HP-1, HP-2, and HP-3, differ by only one amino acid, and it is thought that HP-2 may represent a proteolytic product of HP-1 and/or HP-3. To help determine the relationship of these three proteins, we isolated a nearly full-length cDNA encoding HP-1 with a sequence very similar to, but different from, the previously isolated HP-1 and -3 cDNAs. Gene copy number experiments established that there were at least two but fewer than five defensin genes with a high level of similarity to the HP-1 cDNA (HP-1/3-like). Three genomic clones were isolated that contained two different configurations of the HP-1/3-like sequences. Sequencing established that one encoded the HP-1 peptide, whereas the other encoded HP-3. Analysis of DNAs obtained from 18 unrelated individuals by Southern blot analysis revealed the expected fragments as well as additional fragments that were not present in the genomic clones. This suggested the possibility of alleles; however, when DNAs from families were examined, these fragments did not segregate in an obvious Mendelian fashion. The HP-1/3-like defensin genes are on human chromosome 8. Surprisingly, somatic cell hybrid mapping showed that the number of HP-1/3-like genes on isolated copies of chromosome 8 was variable. We conclude that individuals can inherit versions of chromosome 8 harboring either two or three copies of the genes that encode the HP-1, HP-2, and/or HP-3 peptides.
Corticostatins, defensins, and cryptdins are members of an unusual family of peptides that have been isolated from the alveolar macrophages, neutrophils, bone marrow leukocytes, and/or intestinal Paneth cells of rabbits, guinea pigs, mice, rats, and/or humans (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) . These proteins are small, often cationic peptides (29-35 amino acids) with a characteristic cysteine-rich structure and were originally isolated because of their anti-adrenocorticotrophin hormone (corticostatic) (6, 7) or antimicrobial (defensin) activities(1, 2, 3, 4) . Subsequently, sequence analysis of a cDNA clone revealed that cryptdin, from mouse intestinal Paneth cells, is a family member(13) . Recently, Paneth cell sequences of human origin have also been identified(14, 15) . Presently, the family consists of six human, six rabbit, seven rat, two guinea pig, and six mouse sequences.
Three of the human peptides, HP-1, HP-2, and HP-3, are almost identical in sequence. HP-1 and HP-3 are 30 amino acids long and differ only at the first amino acid, which is Ala in HP-1 and Asp in HP-3. HP-2 is 29 amino acids in length and may simply be a proteolytic product of one or both of the other two peptides as it begins with the second amino acid (Cys) of HP-1 and HP-3. In contrast, sequences of HP-4, HP-5, and HP-6 are very different, sharing only 10 amino acids with each other and the other HP peptides.
The functions of the human peptides have been studied in some detail. At higher concentrations (25-200 µg/ml range), HP-1, HP-2, HP-3, and HP-4 exhibit varying degrees of antimicrobial and/or viricidal properties in vitro(2, 17, 18) . In general, HP-4 seems to have the greatest defensin activity(19) , whereas HP-3 has the least (2) , being completely inert in assays against Candida albicans(18) . At lower concentrations (1-25 µg/ml range), a mixture of HP-1, HP-2, and HP-3 affects mammalian cell growth in that it is cytotoxic against selected cell lines and promotes the growth of others(7, 20, 21, 22) . Curiously, HP-1 and HP-2 are chemotactic for monocytes, whereas HP-3 is not(23) . All three proteins can inhibit protein kinase C in vitro, with HP-2 displaying the greatest inhibitory effect(24) . HP-4 has corticostatic activity, whereas HP-1, HP-2, and HP-3 do not(7, 20) . As HP-5 and HP-6 are known only from their DNA sequences(15, 16) , their functions are unknown; however, it is likely that these peptides have defensin-like activities, similar to their mouse counterparts(10, 12) .
The probable tertiary structure of the peptides has been determined (25) . They are considered to be cyclic as the first and last cysteines are linked(26) . In addition, they form dimers with charged and hydrophobic molecules clustered on two different faces of the molecule, rendering them amphiphilic. Under optimal conditions, HP-1 can permeabilize the outer and inner membranes of Escherichia coli(27) , forming voltage-gated ion channels(28) . Furthermore, the peptides do not display viricidal activity against viruses without envelopes(17) , suggesting that membrane interaction is necessary for defensin-like function. The corticostatin activity of the rabbit peptides occurs via receptor interaction(29, 30) , so it is likely that all of the peptides in this family exert their functions via some type of membrane interaction.
Although much is known about the function and structure of these peptides, relatively little is known about them at the DNA and RNA levels. cDNAs encoding HP-1, HP-3, HP-4, HP-5, and HP-6 have been isolated(15, 16, 31, 32, 33, 34) . The two cDNA sequences encoding HP-1 and HP-3 are almost identical, differing by only three nucleotides; whereas, the cDNA encoding HP-4 shares a 72% identity with the HP-1 and HP-3 cDNAs. The cDNA encoding HP-6 is also very similar to the sequences encoding HP-1, HP-3, and HP-4 in certain regions. For example, nucleotides -9 to +81 of the HP-6 sequence (16) are 84% identical with nucleotides +85 to +174 of the HP-1 sequence(31) . Genes encoding HP-1, HP-3, and HP-5 have also been sequenced(15, 35) , as have two of the rabbit genes (encoding MCP-1 and MCP-2)(36) . Like HP-1, HP-2, and HP-3, the MCP-1 and MCP-2 peptides differ by a single amino acid. Although one might expect that the two rabbit genes are simply alleles of each other, analysis of their gene structure revealed that this is not the case. In contrast, two rat peptides that differ by a single amino acid are apparently allelic(8) . Although two genomic sequences encoding the HP-1 and HP-3 peptides have been identified(35) , it is unclear whether they represent two different genes or two alleles of the same gene.
We originally initiated these studies to determine whether the HP-1, HP-2, and HP-3 peptides are alleles or are encoded by different genes. Gene copy number experiments were performed that determined there is more than one gene per haploid genome. Genomic clones from a lambda phage library were isolated and characterized by restriction digest mapping. The restriction map analysis indicated that two clones were similar to the previously published genomic sequences encoding HP-1 and HP-3(35) , but a third clone was different. Sequencing determined that the former clones encoded HP-1, whereas the third encoded HP-3. To ascertain whether two different alleles or two different genes had been isolated, we performed Southern blot analyses on genomic DNAs from unrelated individuals as well as from families. Novel fragments, which were additional to the ones predicted from the genomic maps, were present in DNAs from unrelated individuals, confirming that there are multiple HP-1/3-like genes. Surprisingly, when DNAs from grandparents, parents, and children within the same family were examined, we discovered that the genes did not segregate in an obvious Mendelian fashion. To determine which fragments corresponded to which gene, DNAs from somatic cell hybrids containing single human chromosome 8s were examined. This analysis determined that the numbers of HP-1/3-like genes on the different chromosome 8s varied. Thus, individuals can inherit unequal numbers of these genes, depending upon which chromosomes they inherit from their parents.
Figure 1: Nucleotide sequence of the pUC4A cDNA encoding HP-1. A plasmid containing a cDNA encoding the 3`-end of HP-1 (pC-A3) was used to isolate a longer cDNA clone from an EMBL phage cDNA library. The insert was subcloned into a pUC vector and sequenced. Numbering of nucleotide positions refers to (31) . Coding regions of the sequence are in capital letters. The part of the coding region corresponding to the mature peptide is underlined. Nucleotides that differ from the previously published HP-1 sequence are in bold italics.
Figure 2:
Estimated gene copy number of the HP-1/3-like defensin genes in the human haploid genome.
Standards (BamHI-digested pC-A3) correspond to gene copy
numbers per haploid genome in 10 µg of genomic DNA. Genomic DNAs
(10 µg) isolated from normal peripheral blood leukocytes and the
peripheral blood leukocytes of a patient with chronic myelogenous
leukemia were digested with EcoRI. All samples were
electrophoresed through 1% agarose gels, transferred to nitrocellulose,
and hybridized with a radiolabeled PstI-SstI fragment
of pC-A3. HindIII-digested lambda DNA and BamHI-digested pC-A3 (4.8 kb) were used as size markers. The
resulting autoradiograms were scanned with a densitometer. Panel
A, representative autoradiogram. Panel B, bar graph
showing results obtained from a scan of the autoradiograph. ,
normal PB; &cjs2100;, CML; &cjs2110;, 1 copy; &cjs2106;, 2 copies;
, 5 copies.
Figure 3: Partial restriction maps of phage DNAs containing sequences corresponding to the HP-1/3-like genes. For simplicity, only pertinent restriction sites are shown. The assignment of the clones' names are based upon the gene sequence. A, Asp718; B, BamHI; Bg, BglI; E, EcoRI; H, HindIII.
Figure 4: Southern blot analysis of the 5`-end of the HP-1/3-like genes in genomic DNAs from unrelated individuals. Genomic DNAs from nine unrelated individuals were digested with Asp718 or BamHI and subjected to Southern blot analysis. To maximize resolution, field inversion gel electrophoresis was used to separate the DNA fragments. The probe was the 1.6-kb EcoRI fragment from the 5`-end of the HP-1 gene (see Fig. 3).
To determine whether the Asp718 bands were indeed alleles, 23 DNAs from a three-generation pedigree were examined by digesting with Asp718 and probing with the pUC4A cDNA. Surprisingly, using this approach we were able to show that the two smaller Asp718 bands were not the products of the larger band, as many family members had only one, rather than both, of the two smaller bands (Fig. 5). Furthermore, the combined data suggested that our probe was probably detecting two (or more) highly related genes with alleles that were comigrating and therefore cohybridizing. An alternative but less likely explanation of the data was that at the DNA level we were detecting a gene (or genes) that did not have a corresponding allele (or alleles) on the sister chromosome. To help resolve the situation, additional experiments were performed using BglI digests of DNAs from several smaller pedigrees. Again, after probing with the 1.6-kb EcoRI genomic fragment from the 5`-end of the gene, we were unable to detect alleles that segregated in a typical Mendelian fashion (data not shown).
Figure 5: Southern blot analysis of family DNAs. Genomic DNAs from the mother, father, and three of their offspring were digested with Asp718 and subjected to Southern blot analysis. The filter was probed with pUC4A, representing the HP-1 cDNA (see Fig. 1). Lanes 1 and 2 contain the parental DNAs, whereas lanes 3-5 contain the childrens' DNAs.
Figure 6: Southern blot analysis of DNAs from somatic cell hybrids containing individual chromosome 8s. DNAs from 16 different somatic cell hybrids containing either one or two human chromosome 8s were digested with BglI and subjected to Southern blot analysis with a 1.6-kb EcoRI fragment from the HP-1 gene (see Fig. 3). This probe does not cross-react with hamster DNA. Hybrids in lanes 1-4, 8-10, 12, and 13 contain the same copy of chromosome 8 from a single individual, whereas the hybrids in lanes 5, 6, and 14-16 contain the other chromosome 8 from that individual. The hybrid in lane 7 contains both copies of chromosome 8 from that individual, whereas the hybrid in lane 11 contains a single chromosome 8 from a different individual. Lanes 1-16 correspond to hybrids 1HL3, 1HL26, 1HL27, 1HL33, 1HL35, 1HL19, 1HL38, 1SHL1, 1SHL12, 1SHL15, 1HL12, 1SHL3, 1SHL27, 1SHL7, 1SHL30, and 1SHL32, respectively(45) .
Our somatic cell hybrid data showed that individuals can inherit copies of chromosome 8 with either two or three highly related loci corresponding to the 5` regions of the HP-1/3-like genes. To confirm that all of these bands actually contained 5`-coding regions in addition to the genomic sequences, the somatic cell hybrid blot was rehybridized with the pUC4A cDNA. As expected, the same bands were detected by pUC4A along with additional bands (data not shown).
We were interested in determining whether the peptides HP-1, HP-2, and HP-3 originate from a single gene or alternatively, whether they represent the products of almost identical, but separate genes. By a combination of studies such as gene copy number experiments and Southern blot analysis, we showed that multiple HP-1/3-like genes exist; however, using this approach we were still unable to distinguish whether the HP-1 and HP-3 peptides arose from separate genes or whether they were allelic variants of a single gene. We reasoned that by analyzing DNAs from somatic cell hybrids containing opposing copies of chromosome 8 from a single individual, we would be able to compare the band patterns on the individual chromosomes and distinguish between the two possibilities. To our surprise, we discovered that the chromosomes harbored unequal numbers of the HP-1/3-like genes, showing that unequal numbers of these genes can be inherited.
Our somatic cell hybrid data demonstrates that the HP-1 and HP-3 sequences we isolated represent two genes rather than two alleles. The 1.2- and 2.9-kb BglI bands in the somatic cell hybrid DNAs (see Fig. 6), found on a single chromosome 8, correspond with the expected HP-1 and HP-3 fragments, respectively, as seen in Fig. 3. The 2.3-kb band, which was present in all the somatic cell hybrid DNAs, corresponds with the expected band from the published HP-1/HP-3 gene sequence (35) and spans the BglI site just 5` of exon I to the BglI site inside exon II. In all of the hybrids, we also detected a small (500-600-base pair) BglI fragment with the genomic probe, which did not hybridize with the cDNA (not shown). This fragment represents the end 5`-BglI site depicted in Fig. 3to the first internal BglI site in HP-1. The most likely origin of the 1.7-kb band detected in the hybrids would be a segment that spans the outer BglI site to the site between exons I and II. It is presently unclear whether the 1.7-kb fragment represents an allele of HP-1 or HP-3 or if it encodes a third HP-1/3-like gene (such as HP-2) or pseudogene.
Given the similarities of the HP-4, HP-5, and HP-6 sequences (15, 16, 34) with the HP-1/3-like sequences in the 5`-transcribed regions, it might seem reasonable to suspect that one of the strongly hybridizing bands we detected represents one of these other genes; however, comparison of our data with the published data shows that this is probably not the case. For example, the genomic EcoRI fragment, which we used as a probe, is 5` to the region of shared similarity with HP-5(15) and is not necessarily present upstream of the HP-5 gene. Still, the EcoRI fragment did detect three other larger, much weaker hybridizing bands in the somatic cell hybrids digested with BglI, which cross-hybridized with the pUC4A cDNA (data not shown) and two smaller (6.0 and 7.0 kb), weaker bands in the BamHI digests (Fig. 4). It is possible that these weaker bands represent minor regions of shared similarity with other genes such as HP-4, HP-5, and HP-6.
Given our inability to demonstrate typical Mendelian inheritance within families combined with our somatic cell hybrid data, we propose that variable numbers of the HP-1/3-like genes are transmitted to individuals within the general population. It is probable that the genes are physically linked on the individual chromosomes, as preliminary data using field inversion gel electrophoresis showed a single hybridizing EagI band of 300 kb by Southern blot analysis (data not shown). Thus, one would inherit a block of genes (most likely two or three) from each parent, the number being determined by the number on the parental chromosome 8. In agreement with our hypothesis is the finding that approximately 10% of the population lacks the HP-3 peptide(47) . Interestingly, the 4.0-kb Asp718 band we observed was present in DNAs from 16 of 18 unrelated individuals (89%). This Asp718 fragment was present in our complete HP-3 genomic clone (data not shown), which had the the 2.9-kb BglI fragment. As the 2.9-kb BglI fragment was a third HP-1/3-like fragment in the somatic cell hybrid, it is intriguing to speculate that this configuration of the HP-3 gene encodes the HP-3 protein present in 90% of the population. In fact, although this was the fragment that did not display the hypothesized Mendelian inheritance when we examined our three-generation pedigree (Fig. 5), analysis of the familial data shows proper segregation when this allele is used as a haplotype marker. Still, there is a problem with our hypothesis since a second genomic sequence encoding HP-3 has been reported(35) , and its role would be left unexplained.
In summary, we detected a second cDNA sequence (pUC4A) that encodes the HP-1 peptide and showed that the numbers of the defensin HP-1/3-like genes vary from individual to individual. Furthermore, we proved that HP-1 and HP-3 are encoded by two different genes on the same chromosome. At present, no sequence has been detected that is known to specifically encode HP-2, and HP-2 may be a proteolytic product of the HP-1 or HP-3 peptides (or both). One interesting possibility is that one of the mRNAs thought to encode HP-1 may actually encode HP-2. For example, subtle differences in the mRNAs might be indicative of cell-specific expression. The final, processed peptide would then reflect the parental cell type in which it was originally transcribed. It is not unrealistic to believe that a specific HP-2 gene exists since we have shown that there can be three configurations of the HP-1/3-like genes on a single chromosome. Further studies will be necessary to elucidate the exact relationship between the genomic structure, expression, and translation of this interesting family of peptides.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X52053[GenBank].