Cardiovascular abnormalities with normal blood pressure in tissue kallikrein-deficient mice

Tissue kallikrein is a serine protease thought to be involved in the generation of bioactive peptide kinins in many organs like the kidneys, colon, salivary glands, pancreas, and blood vessels. Low renal synthesis and urinary excretion of tissue kallikrein have been repeatedly linked to hypertension in animals and humans, but the exact role of the protease in cardiovascular function has not been established largely because of the lack of specific inhibitors. This study demonstrates that mice lacking tissue kallikrein are unable to generate significant levels of kinins in most tissues and develop cardiovascular abnormalities early in adulthood despite normal blood pressure. The heart exhibits septum and posterior wall thinning and a tendency to dilatation resulting in reduced left ventricular mass. Cardiac function estimated in vivo and in vitro is decreased both under basal conditions and in response to βadrenergic stimulation. Furthermore, flow-induced vasodilatation is impaired in isolated perfused carotid arteries, which express, like the heart, low levels of the protease. These data show that tissue kallikrein is the main kinin-generating enzyme in vivo and that a functional kallikrein–kinin system is necessary for normal cardiac and arterial function in the mouse. They suggest that the kallikrein–kinin system could be involved in the development or progression of cardiovascular diseases.

Structure of neurolysin reveals a deep channel that limits substrate access

The zinc metallopeptidase neurolysin is shown by x-ray crystallography to have large structural elements erected over the active site region that allow substrate access only through a deep narrow channel. This architecture accounts for specialization of this neuropeptidase to small bioactive peptide substrates without bulky secondary and tertiary structures. In addition, modeling studies indicate that the length of a substrate N-terminal to the site of hydrolysis is restricted to approximately 10 residues by the limited size of the active site cavity. Some structural elements of neurolysin, including a five-stranded β-sheet and the two active site helices, are conserved with other metallopeptidases. The connecting loop regions of these elements, however, are much extended in neurolysin, and they, together with other open coil elements, line the active site cavity. These potentially flexible elements may account for the ability of the enzyme to cleave a variety of sequences.

The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product.

Tumors express peptide antigens capable of being recognized by tumor-specific cytotoxic T lymphocytes (CTL). Immunization of mice with a carcinogen-induced colorectal tumor, CT26, engineered to secrete granulocyte/macrophage colony-stimulating factor, routinely generated both short-term and long-term CTL lines that not only lysed the parental tumor in vitro, but also cured mice of established tumor following adoptive transfer in vivo. When either short-term or long-term CTL lines were used to screen peptides isolated from CT26, one reverse-phase high performance liquid chromatography peptide fraction consistently sensitized a surrogate target for specific lysis. The bioactivity remained localized within one fraction following multiple purification procedures, indicating that virtually all of the CT26-specific CTL recognized a single peptide. This result contrasts with other tumor systems, where multiple bioactive peptide fractions have been detected. The bioactive peptide was identified as a nonmutated nonamer derived from the envelope protein (gp70) of an endogenous ecotropic murine leukemia provirus. Adoptive transfer with CTL lines specific for this antigen demonstrated that this epitope represents a potent tumor rejection antigen. The selective expression of this antigen in multiple non-viral-induced tumors provides evidence for a unique class of shared immunodominant tumor associated antigens as targets for antitumor immunity.

Chymase cleavage of stem cell factor yields a bioactive, soluble product

Stem cell factor (SCF) is produced by stromal cells as a membrane-bound molecule, which may be proteolytically cleaved at a site close to the membrane to produce a soluble bioactive form. The proteases producing this cleavage are unknown. In this study, we demonstrate that human mast cell chymase, a chymotrypsin-like protease, cleaves SCF at a novel site. Cleavage is at the peptide bond between Phe-158 and Met-159, which are encoded by exon 6 of the SCF gene. This cleavage results in a soluble bioactive product that is 7 amino acids shorter at the C terminus than previously identified soluble SCF. This research shows the identification of a physiologically relevant enzyme that specifically cleaves SCF. Because mast cells express the KIT protein, the receptor for SCF, and respond to SCF by proliferation and degranulation, this observation identifies a possible feedback loop in which chymase released from mast cell secretory granules may solubilize SCF bound to the membrane of surrounding stromal cells. The liberated soluble SCF may in turn stimulate mast cell proliferation and differentiated functions; this loop could contribute to abnormal accumulations of mast cells in the skin and hyperpigmentation at sites of chronic cutaneous inflammation.

Bioactive and nuclease-resistant l-DNA ligand of vasopressin

In vitro selection experiments have produced nucleic acid ligands (aptamers) that bind tightly and specifically to a great variety of target biomolecules. The utility of aptamers is often limited by their vulnerability to nucleases present in biological materials. One way to circumvent this problem is to select an aptamer that binds the enantiomer of the target, then synthesize the enantiomer of the aptamer as a nuclease-insensitive ligand of the normal target. We have so identified a mirror-image single-stranded DNA that binds the peptide hormone vasopressin and have demonstrated its stability to nucleases and its bioactivity as a vasopressin antagonist in cell culture.

Posttranslational modification of the glycosylation inhibiting factor (GIF) gene product generates bioactive GIF

Glycosylation inhibiting factor (GIF) and macrophage migration inhibitory factor (MIF) share an identical structure gene. Here we unravel two steps of posttranslational modifications in GIF/MIF molecules in human suppressor T (Ts) cell hybridomas. Peptide mapping and MS analysis of the affinity-purified GIF from the Ts cells revealed that one modification is cysteinylation at Cys-60, and the other is phosphorylation at Ser-91. Cysteinylated GIF, but not the wild-type GIF/MIF, possessed immunosuppressive effects on the in vitro IgE antibody response and had high affinity for GIF receptors on the T helper hybridoma cells. In vitro treatment of wild-type recombinant human GIF/MIF with cystine resulted in preferential cysteinylation of Cys-60 in the molecules. The cysteinylated recombinant human GIF and the Ts hybridoma-derived cysteinylated GIF were comparable both in the affinity for the receptors and in the immunosuppressive activity. Polyclonal antibodies specific for a stretch of the amino acid sequence in α2-helix of GIF bound bioactive cysteinylated GIF but failed to bind wild-type GIF/MIF. These results strongly suggest that cysteinylation of Cys-60 and consequent conformational changes in the GIF/MIF molecules are responsible for the generation of GIF bioactivity.

The recombinant proregion of transforming growth factor beta1 (latency-associated peptide) inhibits active transforming growth factor beta1 in transgenic mice.

All three isoforms of transforming growth factors beta (TGF-betal, TGF-beta2, and TGF-beta3) are secreted as latent complexes and activated extracellularly, leading to the release of the mature cytokines from their noncovalently associated proregions, also known as latency-associated peptides (LAPs). The LAP region of TGF-beta1 was expressed in a baculovirus expression system and purified to homogeneity. In vitro assays of growth inhibition and gene induction mediated by TGF-beta3 demonstrate that recombinant TGF-beta1 LAP is a potent inhibitor of the activities of TGF-betal, -beta2, and -beta3. Effective dosages of LAP for 50% neutralization of TGF-beta activities range from 4.7- to 80-fold molar excess depending on the TGF-beta isoform and activity examined. Using 125I-labeled LAP, we show that the intraperitoneal application route is effective for systemic administration of LAP. Comparison of concentrations of LAP in tissues shows a homogenous pattern in most organs with the exception of heart and muscle, in which levels of LAP are 4- to 8-fold lower. In transgenic mice with elevated hepatic levels of bioactive TGF-betal, treatment with recombinant LAP completely reverses suppression of the early proliferative response induced by TGF-beta1 in remnant livers after partial hepatectomy. The results suggest that recombinant LAP is a potent inhibitor of bioactive TGF-beta both in vitro and in vivo, after intraperitoneal administration. Recombinant LAP should be a useful tool for novel approaches to study and therapeutically modulate pathophysiological processes mediated by TGF-beta3.

Two alternative processing pathways for a preprohormone: a bioactive form of secretin.

An N-terminally 9-residue elongated form of secretin, secretin-(-9 to 27) amide, was isolated from porcine intestinal tissue and characterized. Current knowledge about peptide processing sites does not allow unambiguous prediction of the signal peptide cleavage site in preprosecretin but suggests cleavage in the region of residues -10 to -14 counted upstream from the N terminus of the hormone. However, the structure of the isolated peptide suggests that the cleavage between the signal peptide and the N-terminal propeptide occurs at the C-terminal side of residue -10. Moreover, the isolated peptide demonstrates that secretin can be fully processed C-terminally prior to the final N-terminal cleavage. The results from this report, and those from earlier studies, where C-terminally elongated variants were isolated, show that the processing of the secretin precursor may proceed by one of two alternative pathways, in which either of the two ends is processed first. The bioactivity of the N-terminally extended peptide on exocrine pancreatic secretion was lower than that of secretin, indicating the importance of the finally processed free N terminus of the hormone for interaction with secretin receptors.

Growth hormone secretagogues: characterization, efficacy, and minimal bioactive conformation.

Another class of growth hormone (GH) secretagogues has been discovered by altering the backbone structure of a flexible linear GH-releasing peptide (GHRP). In vitro and in vivo characterization confirms these GH secretagogues as the most potent and smallest (M(r) < 500) reported. Anabolic efficacy is demonstrated in rodents with intermittent delivery. A convergent model of the bioactive conformation of GHRPs is developed and is supported by the NMR structure of a highly potent cyclic analog of GHRP-2. The model and functional data provide a logical framework for the further design of low-molecular weight secretagogues and illustrate the utility of an interdisciplinary approach to elucidating potential bound-state conformations of flexible peptide ligands.

Aspirin triggers previously undescribed bioactive eicosanoids by human endothelial cell-leukocyte interactions.

Aspirin [acetylsalicylic acid (ASA)], along with its analgesic-antipyretic uses, is now also being considered for cardiovascular protection and treatments in cancer and human immunodeficiency virus infection. Although many of ASA's pharmacological actions are related to its ability to inhibit prostaglandin and thromboxane biosynthesis, some of its beneficial therapeutic effects are not completely understood. Here, ASA triggered transcellular biosynthesis of a previously unrecognized class of eicosanoids during coincubations of human umbilical vein endothelial cells (HUVEC) and neutrophils [polymorphonuclear leukocytes (PMN)]. These eicosanoids were generated with ASA but not by indomethacin, salicylate, or dexamethasone. Formation was enhanced by cytokines (interleukin 1 beta) that induced the appearance of prostaglandin G/H synthase 2 (PGHS-2) but not 15-lipoxygenase, which initiates their biosynthesis from arachidonic acid in HUVEC. Costimulation of HUVEC/PMN by either thrombin plus the chemotactic peptide fMet-Leu-Phe or phorbol 12-myristate 13-acetate or ionophore A23187 leads to the production of these eicosanoids from endogenous sources. Four of these eicosanoids were also produced when PMN were exposed to 15R-HETE [(15R)-15-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid] and an agonist. Physical methods showed that the class consists of four tetraene-containing products from arachidonic acid that proved to be 15R-epimers of lipoxins. Two of these compounds (III and IV) were potent inhibitors of leukotriene B4-mediated PMN adhesion to HUVEC, with compound IV [(5S,6R,15R)-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoi c acid; 15-epilipoxin A4] active in the nanomolar range. These results demonstrate that ASA evokes a unique class of eicosanoids formed by acetylated PGHS-2 and 5-lipoxygenase interactions, which may contribute to the therapeutic impact of this drug. Moreover, they provide an example of a drug's ability to pirate endogenous biosynthetic mechanisms to trigger new mediators.

Association of the "major histocompatibility complex subregion" I-J determinant with bioactive glycosylation-inhibiting factor.

Murine suppressor T-cell hybridoma cells (231F1) secrete not only bioactive glycosylation-inhibiting factor (GIF) but also an inactive peptide comparable to bioactive GIF peptide in its molecular size and reactivity with anti-GIF; the amino acid sequence of the inactive peptide is identical to that of the bioactive homologue. The inactive GIF peptide in culture supernatant of both the 231F1 cells and a stable transfectant of human GIF cDNA in the murine suppressor T hybridoma selectively bound to Affi-Gel 10, whereas bioactive GIF peptides from the same sources failed to bind to the gel. The inactive cytosolic human GIF from the stable transfectant and Escherichia coli-derived recombinant human GIF also had affinity for Affi-Gel 10. Both the bioactive murine GIF peptide from the suppressor T hybridoma and bioactive recombinant human GIF from the stable transfectant bound to the anti-I-J monoclonal antibody H6 coupled to Affi-Gel. However, bioactive hGIF produced by a stable transfectant of human GIF cDNA in BMT10 cells failed to be retained in H6-coupled Affi-Gel. These results indicate that the I-J specificity is determined by the cell source of the GIF peptide and that the I-J determinant recognized by monoclonal antibody H6 does not represent a part of the primary amino acid sequence of GIF. It appears that the epitope is generated by a posttranslational modification of the peptide.