Peptide localization and gene structure of cryptdin 4, a differentially expressed mouse paneth cell alpha-defensin.

Paneth cells in crypts of the small intestine express antimicrobial peptides, including alpha-defensins, termed cryptdins in mice. Of the known Paneth cell alpha-defensins, the cryptdin 4 gene is unique, because it is inactive in the duodenum and expressed at maximal levels in the distal small bowel (D. Darmoul and A. J. Ouellette, Am. J. Physiol. 271:G68-G74, 1996). With a cryptdin 4-specific antibody, immunohistochemical staining of ileal Paneth cells was strong and specific for cytoplasmic granules, demonstrating that this microbicidal peptide is a secretory product of Paneth cells in the distal small intestine. Consistent with the pattern of cryptdin 4 mRNA distribution along the length of the gut, the cryptdin 4 peptide was not detected in duodenum. Structurally, the cryptdin 4 gene resembles other Paneth cell alpha-defensin genes. Its two exons, transcriptional start site, intron, splice sites, and 3' flanking sequences are characteristic of the highly conserved mouse alpha-defensin genes. However, in the region upstream of the transcriptional initiation site, the cryptdin 4 gene contains a repeated 130-bp element that is unique to this alpha-defensin gene. Every independent cryptdin 4 genomic clone examined carries the repeated element, which contains putative recognition sequences for TF-IID-EIIA, cMyc-RS-1, and IgHC.2/CuE1.1; the repeat proximal to the start of transcription replaces DNA at the corresponding position in other mouse alpha-defensin genes. We speculate that this unique duplicated element may have a cis-acting regulatory role in the positional specificity of cryptdin 4 gene expression.

Differences in the concentrations of small, anionic, antimicrobial peptides in bronchoalveolar lavage fluid and in respiratory epithelia of patients with and without cystic fibrosis.

Affinity-purified rabbit polyclonal (PAB96-1) and mouse monoclonal (1G9-1C2) antibodies to synthetic H-DDDDDDD-OH, an antimicrobial anionic peptide (AP) originally isolated from ovine pulmonary surfactant, were prepared and used to assess the concentrations of AP-like molecules in human respiratory tract samples. In bronchoalveolar lavage fluids, concentrations of AP-like molecules measured by enzyme-linked immunosorbent assay were significantly lower in 13 patients with cystic fibrosis (CF) (mean +/- standard deviation [SD], 0.78 +/- 0.46 mM) than in 34 patients without CF (1. 30 +/- 0.66 mM) (P = 0.01). In pulmonary tissues of three patients without CF, very little antigen was stained in the apical cytoplasm of the bronchial and bronchiolar epithelium yet robust staining was seen in the alveolar epithelium. In pulmonary tissues of three patients with CF, robust staining of antigen was seen in the apical cytoplasm of the bronchial and bronchiolar epithelium yet no staining was seen in the alveolar epithelium. These results show that AP-like molecules are present in healthy human respiratory tract samples and differ in concentration and location of expression in patients with and without CF.

Antimicrobial peptides as mediators of epithelial host defense.

Mammalian epithelial surfaces are remarkable for their ability to provide critical physiologic functions in the face of frequent microbial challenges. The fact that these mucosal surfaces remain infection-free in the normal host suggests that highly effective mechanisms of host defense have evolved to protect these environmentally exposed tissues. Throughout the animal and plant kingdoms, endogenous genetically encoded antimicrobial peptides have been shown to be key elements in the response to epithelial compromise and microbial invasion. In mammals, a variety of such peptides have been identified, including the well-characterized defensins and cathelicidins. A major source of these host defense molecules is circulating phagocytic leukocytes. However, more recently, it has been shown that resident epithelial cells of the skin and respiratory, alimentary, and genitourinary tracts also synthesize and release antimicrobial peptides. Both in vitro and in vivo data support the hypothesis that these molecules are important contributors to intrinsic mucosal immunity. Alterations in their level of expression or biologic activity can predispose the organism to microbial infection. The regulatory and developmental aspects of antimicrobial peptide synthesis are discussed from a perspective that emphasizes the possible relevance to pediatric medicine.

Detection of anionic antimicrobial peptides in ovine bronchoalveolar lavage fluid and respiratory epithelium.

Three small antimicrobial anionic peptides (AP) were originally isolated from an ovine pulmonary surfactant. However, their presence in bronchoalveolar lavage (BAL) fluid and tissues of the respiratory tract is unknown. In this study, we made affinity-purified rabbit polyclonal and mouse monoclonal antibodies to synthetic H-DDDDDDD-OH. Antibody specificity was assessed by a competitive enzyme-linked immunosorbent assay (ELISA), and the exact epitope binding sites were determined with analog peptides synthesized on derivatized cellulose. These antibodies were used to detect AP in BAL fluid by ELISA and in respiratory tissues by Western blot analysis and immunocytochemistry. BAL fluid from 25 sheep contained 0.83 +/- 0.33 mM AP (mean +/- standard deviation; range, 0.10 to 1.59 mM) and was antimicrobial. The presence of AP in BAL fluid was confirmed by reverse-phase high-pressure liquid chromatography fractionation followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry on those fractions which were positive by competitive ELISA and demonstrated antimicrobial activity. In Western blots, polyclonal antibody PAB96-1 and monoclonal antibody 1G9-1C2 (5.0 micrograms/ml) detected four bands in solubilized turbinate and tracheal epithelial cells (53.7, 31.2, 28.0, and 25.7 kDa) and five bands in lung homogenates (53.5, 37.1, 31.2, 28.0, and 25.7 kDa). Only a single band was seen in solubilized liver and small-intestine homogenates, and no bands were seen in blots containing BAL fluid, albumin, or kidney or spleen homogenates. In pulmonary-tissue sections, both antibodies PAB96-1 and 1G9-1C2 identified accumulated protein in the apical cytoplasm of the bronchial and bronchiolar epithelia, in the cytoplasm of pulmonary endothelial cells, and in an occasional alveolar macrophage. As a first step in identifying a candidate AP precursor gene(s), degenerate oligonucleotides representing all possible coding combinations for H-GADDDDD-OH and H-DDDDDDD-OH were synthesized and used to probe Southern blots of sheep genomic DNA. Following low-stringency washes and a 2-day exposure, strongly hybridizing bands could be identified. One degenerate oligonucleotide, SH87, was used as a hybridization probe to screen a sheep phage genomic library. Two independent phage contained the H-GADDDDD-OH coding sequence as part of a larger predicted protein. AP may originate as part of an intracellular precursor protein, with multistep processing leading to the release of the heptapeptide into mucosal secretions. There it may interact with other innate pulmonary defenses to prevent microbial infection.

Antimicrobial peptides: an emerging concept in cutaneous biology.

Antimicrobial peptides are part of the host defense systems of plants, insects, fish, amphibia, birds, and mammals. These small proteins were previously thought of as an evolutionarily ancient system of immune protection with little relevance to the normal function of human skin. Recent developments have found that mammalian skin expresses these gene-encoded peptide antibiotics during inflammatory events such as wound repair, contact dermatitis, and psoriasis. The presence of these peptides in the skin forms a barrier for innate host protection against microbial pathogenesis. Furthermore, antimicrobial peptides also act on animal cells by stimulating them to change behaviors such as syndecan expression, chemotaxis, and chloride secretion. The combination of effects on host cells with antimicrobial action in a single molecule represents an efficient defense and response system against injury. Understanding the action of antimicrobial peptides in skin may yield further insight into the mechanism of innate cutaneous disease control and provide new approaches to therapy of wounds and inflammatory dermatitis.

Antimicrobial peptide expression is developmentally regulated in the ovine gastrointestinal tract.

Antimicrobial peptides are abundant components of the innate immune system present in species throughout the plant and animal kingdoms. In mammals, these immune peptides have been localized to epithelial tissues of the pig, mouse, rat, cow and human gastrointestinal tracts. We have identified in sheep two members of the beta-defensin antimicrobial peptide gene family that are expressed in a unique pattern throughout the gastrointestinal tract. Sheep beta-defensin 1 mRNA is the most prevalent from tongue to colon with the exception of the distal ileum, where beta-defensin 2 mRNA predominates. Sheep beta-defensin expression varies significantly between animals and is developmentally regulated both pre- and postnatally. These changes in antimicrobial peptide expression may correlate with anatomical differentiation as well as physiologic adaptations to extra-uterine life.

Localization and genomic organization of sheep antimicrobial peptide genes.

Antimicrobial peptides are an abundant and diverse component of animal innate immunity. Within mammalian species, defensins and cathelicidins are the two principal antimicrobial peptide families. We identified and sequenced ten new sheep genes which encode potential antimicrobial peptides including two beta-defensins and eight cathelicidins. We mapped the two-exon beta-defensin genes to sheep chromosome 26 and the four-exon cathelicidin genes to sheep chromosome 19 using sheep-hamster somatic cell hybrids in conjunction with flow-sorted sheep chromosomes. These assignments confirm homology between sheep, cattle, mouse, and human antimicrobial peptide gene families. Contig construction for the sheep cathelicidin gene family demonstrates that three genes, OaDodeA, OaDodeB, and OaMAP-34, are present head-to-tail in a 14.5 kb region, and that four proline/arginine-rich genes, OaBac5, OaBac7.5, OaBac11, and OaBac6, are arranged head-to-tail in a region covering 30.5 kb. This richly diverse family of sheep cathelicidin peptides is encoded in a gene array which may reflect the mechanism of its evolution.

The mouse genome encodes a single homolog of the antimicrobial peptide human beta-defensin 1.

The cysteine-rich beta-defensin peptides are broad-spectrum bactericidal agents expressed in epithelial and myeloid tissues. The human beta-defensin-1 (hBD-1) gene maps adjacent to the human alpha-defensin cluster and is expressed in the respiratory, gastrointestinal and genitourinary tracts. Here, we characterize a mouse beta-defensin gene (mBD-1) which is: (1) closely related to hBD-1 both in sequence and gene organization; (2) expressed at high levels in the mouse kidney and at lower levels in brain, heart, lung, uterus, spleen, skeletal muscle, stomach, and small intestine; and (3) maps to mouse chromosome 8 at or near the location of the mouse alpha-defensin genes. These data indicate that mBD-1 is a close homolog of hBD-1, and suggest that analysis of its role in mouse host defense may provide significant insights into human epithelial innate immunity.

Small, anionic, and charge-neutralizing propeptide fragments of zymogens are antimicrobial.

Some inactive precursor proteins, or zymogens, contain small, amino terminus, homopolymeric regions of Asp that neutralize the cationic charge of the active protein during synthesis. After posttranslational cleavage, the anionic propeptide fragment may exhibit antimicrobial activity. To demonstrate this, ovine trypsinogen activation peptide, and frog (Xenopus laevis) PYL activation peptide, both containing homopolymeric regions of Asp, were synthesized and tested against previously described surfactant-associated anionic peptide. Peptides inhibited the growth of both gram-negative (MIC, 0.08 to 3.00 mM) and gram-positive (MIC, 0.94 to 2.67 mM) bacteria. Small, anionic, and charge-neutralizing propeptide fragments of zymogens form a new class of host-derived antimicrobial peptides important in innate defense.

Molecular analysis of the sheep cathelin family reveals a novel antimicrobial peptide.

Cathelin-related genes are characterized by the presence of a prepro sequence which is highly conserved both within and between species. 3' RACE analysis on sheep bone marrow RNA, using a primer based on a conserved cathelin family coding region, demonstrated the presence of at least three ovine cathelin-related cDNAs. One of these encodes a novel prepropeptide with a predicted C-terminal cleavage product RGLRRLGRKIAHG-VKKYGPTVLRIIRIAG. The chemically synthesized form of this 29 amino acid peptide is shown to be a thermostable, broad spectrum, bactericidal agent.

Mouse Paneth cell defensins: primary structures and antibacterial activities of numerous cryptdin isoforms.

Cryptdins are antimicrobial peptides of the defensin family that are produced by intestinal Paneth cells. mRNAs encoding 17 cryptdin isoforms have been characterized from a cDNA library generated from a single jejunal crypt. Six cryptdin cDNAs correspond to known peptides, and the remainder predict 11 novel Paneth cell defensins. Most cryptdin cDNAs have > or = 93% nucleotide sequence identity overall, except for cryptdin 4 and 5 cDNAs, whose respective mature peptide-encoding regions are only 74 and 78% identical to that of cryptdin 1. Cryptdin cDNAs differ at a small number of nucleotide positions: frequent substitutions were found in codons 38 and 52 of the propiece and in codons 68, 73, 76, 87, and 89 of the deduced peptides; cDNA clones with changes in codons 74, 83, and 88 were found, but there were fewer of these. The antimicrobial activities of cryptdins 1 to 6 were tested against Escherichia coli ML35 in two assays. In an agar diffusion assay, the potencies of cryptdins 1 to 3, 5, and 6 were approximately equivalent to that of rabbit neutrophil defensin NP-1 but cryptdin 4 was 30 times more active than NP-1. In a bactericidal assay system, cryptdins 1 and 3 to 6 were equally active at 10 micrograms/ml but cryptdin 2 and rabbit NP-1 were not active at this concentration. Since cryptdins 2 and 3 differ only at residue 10 (Thr and Lys, respectively), this amino acid appears to function in bactericidal interaction with E. coli. The demonstration that Paneth cells express a diverse population of microbicidal defensins further implicates cryptdins in restricting colonization or invasion of small intestinal epithelium by bacteria.

A family of defensin-like genes codes for diverse cysteine-rich peptides in mouse Paneth cells.

Cryptdins constitute a diverse population of defensins in Paneth cells of intestinal crypts. In mice, certain intestinal mRNAs, termed "CRSIC" and "CRS4C," are considered to be cryptdin-related sequences, because their prepro-coding sequences are 94% identical to those of cryptdin-1 mRNA; however, their predicted products, which are cationic, cysteine-rich peptides are not defensins (A. J. Ouellette and J. C. Lualdi, J. Biol. Chem. 265: 9831-9837, 1990). Here we describe several mouse small intestinal mRNAs and genes that code for CRS4C prepropeptides. The 10-kDa deduced CRS4C proteins consist of a prepro sequence, potential monobasic or dibasic peptide cleavage sites, a predicted 3.7-kDa peptide that contains 7 [C]-[X]-[Y] repeats, and a C(N/K)CNPK carboxyl-terminal consensus sequence. In situ hybridization experiments showed that CRS4C mRNAs are found in Paneth cells of adult small bowel. The CRS4C genes closely resemble cryptdin genes, having a two-exon structure with highly conserved transcription start sites, intron-exon junctions, and a single intron of approximately 550 bp. Like the cryptdin genes, exon 1 of CRS4C genes consists of 5' untranslated sequences (UTS) and the prepro-coding region, and exon 2 codes for the predicted mature peptide and 3' UTS. Despite the similarity of first exons in CRS4C and cryptdin genes, their introns exhibit very little homology, and their second exons code for unrelated peptides. Analysis of introns suggests that the ancestral cryptdin and CRS4C genes may have diverged from a common ancestor in the distant past and expanded only recently. We speculate that the cryptdin/CRS genes evolved so that prepro regions encoded by exon 1 were conserved to allow the varied peptides coded by exon 2 to be directed into Paneth cell secretory granules.

Structure and diversity of the murine cryptdin gene family.

Cryptdins are antimicrobial peptides of the defensin family produced by mouse intestinal Paneth cells. Characterization of genomic and cDNA clones of cryptdins 1-3, 5, and 6 revealed that each of these genes has a two-exon structure. The prepro- and mature peptide coding regions are found on different exons separated by an intron of approximately 550 bp. The 5' ends of cryptdin mRNAs are distinguished by a 45-nucleotide untranslated sequence (UTS) encoded completely by the first exon. This feature contrasts with the extended 5' UTS of myeloid defensin mRNAs, which are coded by a third exon that appears to be unique to defensin genes expressed in hematopoietic cells. Sequencing of cryptdin cDNAs from both C3H/HeJ and 129/SVJ mouse small intestine demonstrated the presence of at least 16 different mRNAs, identifying cryptdins as the largest known defensin family. Amplification of these two-exon crypt defensin genes, followed by mutation-induced divergence at a limited number of positions, may have played an important role in the development of a broad-spectrum enteric defense system in the mouse.

Flagellar and acrosomal abnormalities associated with testicular HSV-tk expression in the mouse.

Expression of herpes simplex virus thymidine kinase (HSV-tk) in transgenic mouse testis is associated with abnormalities in spermatogenesis leading to infertility. Our studies of this phenomenon in two transgenic lines led to the identification of a genetic locus that reduced testicular HSV-tk activity and restored fertility. Using light and electron microscopy, we examined spermatogenesis in the infertile transgenic males as well as in the fertile revertants. Infertile males from line 21OH1 had high levels of testicular HSV-tk activity, acrosomal aberrations, and a developmental arrest in spermatogenesis. Infertile males from line ANF1 had lower levels of testicular HSV-tk expression and demonstrated a unique set of structural changes present in the neck and flagellum of epididymal sperm. Revertant ANF1 males, with a significant decrease in testicular HSV-tk expression and a restoration of fertility, showed a marked reduction in the number of sperm abnormalities. Several of the ANF1-specific abnormalities were similar to lesions reported in the sperm of mouse t locus mutants, mouse wobbler homozygotes, and bulls with the Dag-defect.

A novel peptide-producing cell in Xenopus: multinucleated gastric mucosal cell strikingly similar to the granular gland of the skin.

We have characterized a novel peptide-containing cell within the gastric mucosa of Xenopus laevis. The cell is a spherical, multinucleated syncytial structure containing a cytoplasmic space filled with dense rice-shaped granules, and is strikingly similar in morphology to the well-studied granular gland of the amphibian skin. Immunohistochemical and immunogold methods were used to demonstrate that several peptides previously isolated from the granular glands of the skin, including the antimicrobial peptides magainin and PGLa (a peptide with amino-terminal glycine and carboxy-terminal leucinamide), are also stored in granules present in these enteric cells. These data demonstrate that this enteric peptide-producing cell is strikingly similar both morphologically and biochemically to the granular gland, previously considered a highly specialized structure of the amphibian integument. This novel gastric mucosal cell, which we have designated a "granular multinucleated cell," is distinct in its morphology and its diversity of stored peptide products from other well-characterized peptide-containing cells in the vertebrate gastrointestinal tract.

DNA-mediated gene transfer without carrier DNA.

DNA-mediated gene transfer is a procedure which uses purified DNA to introduce new genetic elements into cells in culture. The standard DNA-mediated gene transfer procedure involves the use of whole cell DNA as carrier DNA for the transfer. We have modified the standard DNA-mediated gene transfer procedure to transfer the Herpes simplex virus type 1 thymidine kinase gene (TK) into TK- murine recipient cells in the absence of whole cell carrier DNA. The majority (8/10) of carrier-free transformant lines expressed the TK+ phenotype stably, in sharp contrast to our results with carrier-containing DNA-mediated gene transfer. There was a wide range in donor DNA content among independent transformants. Further analysis on one transformant line using DNA restriction digests and in situ hybridization provided evidence that, in the absence of whole cell carrier DNA, multiple donor DNA sequences became integrated at a single chromosomal site.

Deoxyribonucleic acid-mediated gene transfer in mammalian cells: molecular analysis of unstable transformants and their progression to stability.

To elucidate mechanisms involved in deoxyribonucleic acid-mediated gene transfer, we transferred the herpes simplex virus thymidine kinase gene (TK) into mouse Ltk- cells. Independent TK+ clones (transformants) and derivatives of each were tested for phenotypic expression and the presence and arrangement of TK sequences. Initially, transformants expressed viral TK unstable, with 10% of the cells in each generation losing both the TK+ phenotype and virally derived TK sequences. After a prolonged period in culture, stable subpopulations arose from which the TK+ phenotype and viral sequences were no longer lost at detectable frequency. Analysis of unstable cell populations indicated that individual viral deoxyribonucleic acid molecules were reduced in size, but were linked to other deoxyribonucleic acid to form molecules large enough to be precipitated in a Hirt fractionation. We term these molecules transgenomes. Analysis of independent unstable subclones derived from the primary transformants demonstrated that individual transgenomes could contain multiple copies of the viral TK sequences. Recipient cell lines frequently possessed more than one type of transgenome and possibly multiple copies per cell of each type. Stable derivatives possessed only one of the transgenomes present in the unstable parent, and these sequences were associated with a recipient cell chromosome.

DNA-mediated gene transfer of a circular plasmid into murine cells.

We have used DNA-mediated gene transfer to introduce a recombinant plasmid containing the human beta-globin gene (H beta 1) into cells of a mouse tissue culture line, Ltk-. DNA isolated from independent transfer lines was analyzed by restriction endonuclease digestion, gel electrophoresis, modified Southern blotting, and filter hybridization using H beta 1 as a probe. H beta 1 sequences were present in 80% of the lines at 1-30 copies per cell. Many of the lines gave a hybridization pattern indicative of H beta 1 sequences integrated into high molecular weight DNA. DNA from three cell lines, digested with several restriction enzymes, produced a pattern providing evidence for the presence of circular H beta 1 molecules in the murine recipient cells.

Molecular analysis of chromosome-mediated gene transfer.

Metaphase chromosomes isolated from a cell line carrying the thymidine kinase (TK) gene of herpes simplex virus type I were used to transform the TK-deficient cell line LMTK- to the TK+ phenotype. Four independent transformants were isolated, all of which expressed virus-specific TK. Each of the four transformant cell lines initially became TK- at a rate of 12% per day. All four transformants possessed multiple copies of the TK gene and in one of the four a rearrangement occurred adjacent to the TK sequences. Stable TK+ derivatives of each line, isolated after prolonged cultivation, retained fewer copies of the TK gene than did their unstable parents. The transferred chromosomal fragment was larger than 17 kilobases in each line.