Human α-defensin 5 (HD5, HD5ox to specify the oxidized and disulfide linked form) is a 32-residue cysteine-rich host-defense peptide, expressed and released by small intestinal Paneth cells, that exhibits antibacterial activity against a number of Gram-negative and –positive bacterial strains. To ascertain the contributions of its disulfide array to structure, antimicrobial activity, and proteolytic stability, a series of HD5 double mutant peptides where pairs of cysteine residues corresponding to native disulfide linkages (Cys3—Cys31, Cys5—Cys20, Cys10—Cys30) were mutated to Ser or Ala residues were overexpressed in E. coli, purified and characterized. A hexa mutant peptide, HD5[Serhexa], where all six native Cys residues are replaced by Ser residues was also evaluated. Removal of a single native S—S linkage influences oxidative folding and regioisomerization, antibacterial activity, Gram-negative bacterial membrane permeabilization, and proteolytic stability. Whereas the majority of the HD5 mutant peptides show low-micromolar activity against Gram-negative E. coli ATCC 25922 in colony counting assays, the wild-type disulfide array is essential for low-micromolar activity against Gram-positive S. aureus ATCC 25923. Removal of a single disulfide bond attenuates the activity observed for HD5ox against this Gram-positive bacterial strain. This observation supports the notion that the HD5ox mechanism of antibacterial action differs for Gram-negative and Gram-positive species (Wei, G.; de Leeuw, E., Pazgier, M., Yuan, W., Zou, G., Wang, J., Ericksen, B., Lu, W.-Y.; Lehrer, R. I.; Lu, W. (2009) J. Biol. Chem. 284, 29180-29192), and that the native disulfide array is a requirement for its activity against S. aureus.

Dystrophic cardiac calcification (DCC) is an autosomal recessive trait characterized by calcium phosphate deposits in myocardial tissue. The Abcc6 gene locus was recently found to mediate DCC; however, at the molecular level the causative variants remain to be determined. Examining the sequences of Abcc6 cDNA in DCC-resistant C57BL/6 and DCC-susceptible C3H/He mice, we identified a missense mutation (Cys to Thr at codon 619, rs32756904) at the 3-border of exon 14 that creates an additional donor splice site (GT). Accordingly, an alternative transcript variant was detected, lacking the last 5 bp of exon 14 (-AGG(C/T)GCTgtga-) in DCC-susceptible C3H/He mice that carry the Thr allele. The 5-bp deletion was found to result in premature termination at codon 684, in turn leading to protein deficiency in DCC-susceptible mouse tissue as well as in cells transfected with Abcc6 cDNA lacking the last 5 bp of exon 14. All mouse strains that were found to carry the Thr allele, including C3H/He, DBA/2J, and 129S1/SvJ, were also found to be positive for DCC. In summary, we identified a splice variant leading to a 5-bp deletion in the Abcc6 transcript that gives rise to protein deficiency both in vivo and in vitro. The fact that all mouse strains that carry the deletion also develop dystrophic calcifications further suggests that the underlying splice variant affects the biological function of MRP6 protein and is a cause of DCC in mice.

Dystrophic cardiac calcinosis (DCC) occurs among certain inbred strains of mice and involves necrosis and subsequent calcification as response of myocardial tissue to injury. Using a complete linkage map approach, we investigated the genetics of DCC in an F(2) intercross of resistant C57BL/6J and susceptible C3H/HeJ inbred strains and identified previously a major predisposing quantitative trait locus (QTL), Dyscalc1, on proximal chromosome 7. Analysis of inheritance suggested, however, that DCC is influenced by additional modifier QTL, which have as yet not been mapped. Here, we report the identification by composite interval mapping of the DCC loci Dyscalc2, Dyscalc3, and Dyscalc4 on chromosomes 4, 12 and 14, respectively. Together, the four Dyscalc loci explained 47% of the phenotypic variance of DCC, which was induced by a high-fat diet. Additive epistasis between Dyscalc1 and Dyscalc2 enhanced DCC. Examining recombinant inbred strains, we propose a 10-cM interval containing Dyscalc1 and discuss potential candidate genes.

We performed comprehensive structure–activity relationship (SAR) studies on the peptide portion of antiproliferative factor (APF), a sialylated frizzled-8 related glycopeptide that inhibits normal bladder epithelial and urothelial carcinoma cell proliferation. Glycopeptide derivatives were synthesized by solid-phase methods using standard Fmoc chemistry and purified by RP-HPLC; all intermediate and final products were verified by HPLC-MS and NMR analyses. Antiproliferative activity of each derivative was determined by inhibition of 3H-thymidine incorporation in primary normal human bladder epithelial cells. Structural components of the peptide segment of APF that proved to be important for biological activity included the presence of at least eight of the nine N-terminal amino acids, a negative charge in the C-terminal amino acid, a free amino group at the N-terminus, maintenance of a specific amino acid sequence in the C-terminal tail, and trans conformation for the peptide bonds. These data provide critical guidelines for optimization of structure in design of APF analogues as potential therapeutic agents.