A nonpolymorphic major histocompatibility complex class Ib molecule binds a large array of diverse self-peptides.

Unlike the highly polymorphic major histocompatibility complex (MHC) class Ia molecules, which present a wide variety of peptides to T cells, it is generally assumed that the nonpolymorphic MHC class Ib molecules may have evolved to function as highly specialized receptors for the presentation of structurally unique peptides. However, a thorough biochemical analysis of one class Ib molecule, the soluble isoform of Qa-2 antigen (H-2SQ7b), has revealed that it binds a diverse array of structurally similar peptides derived from intracellular proteins in much the same manner as the classical antigen-presenting molecules. Specifically, we find that SQ7b molecules are heterodimers of heavy and light chains complexed with nonameric peptides in a 1:1:1 ratio. These peptides contain a conserved hydrophobic residue at the COOH terminus and a combination of one or more conserved residue(s) at P7 (histidine), P2 (glutamine/leucine), and/or P3 (leucine/asparagine) as anchors for binding SQ7b. 2 of 18 sequenced peptides matched cytosolic proteins (cofilin and L19 ribosomal protein), suggesting an intracellular source of the SQ7b ligands. Minimal estimates of the peptide repertoire revealed that at least 200 different naturally processed self-peptides can bind SQ7b molecules. Since Qa-2 molecules associate with a diverse array of peptides, we suggest that they function as effective presenting molecules of endogenously synthesized proteins like the class Ia molecules.

A neuroendocrine marker in tissues of the immune system.

Antibodies to chromogranin, a secretory protein marker for the diffuse neuroendocrine system, were used to analyze rat lymphoreticular tissues by means of immunochemistry and immunohistochemistry. Chromogranin-positive cells were present in spleen, lymph node, thymus, and fetal liver. When these organs were gently dispersed and separated on a Ficoll gradient, the chromogranin-immunoreactive cells became enriched in the dense red-cell pellets. The unexpected distribution of these neuroendocrine cells in all immunologically relevant structures suggests that they may link the nervous and immunological systems.

Evaluation of indirect biomarkers for detecting corticosteroids used as illegal growth promoters in beef cattle.

The histological status of the thymus, blood cortisol concentration and circulating neutrophil:lymphocyte ratio were evaluated in 349 slaughtered beef cattle, to assess the potential of these parameters as indirect biomarkers of the illegal use of corticosteroids in meat production. The livers of 20 of the animals were analysed chemically for residues of corticosteroids. The morphology of the thymus was examined for adipose tissue infiltration, cortical atrophy and 'starry sky' appearance, and on the basis of these characteristics, the animals were considered to be negative, suspected or positive for illegal corticosteroid treatment. The animals considered to be negative had a mean cortisol concentration that was significantly higher (29 ng/ml) than that of the animals suspected for corticosteroid treatment (22 ng/ml). Using the chemical analysis as the gold standard for identifying illegally treated animals, the histological examination of the thymus had a sensitivity of 100 per cent and a specificity of 85 per cent. The samples that were positive by chemical analysis had cortisol concentrations of less than 2.0 ng/ml, whereas the mean cortisol concentration of the negative samples was 10.3 ng/ml.

Lymphoproliferative responses to human papillomavirus (HPV) type 16 proteins E6 and E7: outcome of HPV infection and associated neoplasia.

BACKGROUND: Infection with human papillomavirus (HPV) type 16 (HPV16) is a major cause of high-grade cervical intraepithelial neoplasia (CIN). Experiments were planned to evaluate the role of cell-mediated immunity (e.g., lymphocyte proliferation) against HPV in the natural history of HPV-associated neoplasia and to identify antigenic sequences of the HPV16 proteins E6 and E7 against which an immune response may confer protection. METHODS: Forty-nine women with abnormal cervical cytology and biopsy-confirmed CIN were followed through one or more clinic visits. Lymphoproliferative responses of peripheral blood mononuclear cells to HPV16 E6 and E7 peptides were assessed in long-term (3-week) cultures. HPV DNA was detected in cervicovaginal lavage by means of polymerase chain reaction and Southern blotting. Disease status was determined by cervical cytologic examination and colposcopy. Reported P values are two-sided. RESULTS: Subjects with positive lymphoproliferative responses to E6 and/or E7 peptides were more likely to be HPV negative at the same clinic visit than were nonresponders (P = .039). Subjects who were negative for HPV and those with a low viral load were more likely to be responders than were those with a high viral load (P for trend = .037). Responses to N-terminal E6 peptide 369 were associated with absence of HPV infection at the same clinic visit (P = .015). Subjects with positive responses to E6 or E7 peptides at one clinic visit were 4.4 times more likely to be HPV negative at the next visit than were nonresponders (P = .142). Responses to E6 peptide 369 and/or E7 C-terminal peptide 109 were associated with an absence of HPV infection (P = .02 for both) and an absence of CIN (P = .04 and .02, respectively) at the next visit. CONCLUSIONS: Lymphoproliferative responses to specific HPV16 E6 and E7 peptides appear to be associated with the clearance of HPV infection and the regression of CIN.

Chromogranin A-processing proteinases in purified chromaffin granules: contaminants or endogenous enzymes?

It was the purpose of this study to define the chromogranin A-processing proteinases present in highly purified preparations of bovine chromaffin granules. The most active enzyme had a pH optimum of 5.0 and was inhibited by pepstatin. It could be identified immunologically as a cathepsin D-like enzyme and subcellular fractionation established its lysosomal origin. After removal of this enzyme the remaining activity at pH 5.0 was mainly due to a cathepsin B-like proteinase. The presence of this enzyme could also be attributed to lysosomal contamination. In the presence of calcium, a further proteolytic activity became apparent at pH 5.0. This enzyme which was inhibited by rho-chloromercuriphenylsulfonic acid was localized in chromaffin granules. A trypsin-like peptidase, most active at pH 8.2, was enriched in a membrane wash of chromaffin granules. Subcellular fractionation indicated that this enzyme is preferentially bound to the membranes of very dense particles probably representing a subpopulation of chromaffin granules. This study establishes that the most active chromogranin A-degrading proteinases present in highly purified chromaffin granules are attributable to lysosomal contamination. Two enzymes with low activity (a Ca2+ activated proteinase and a trypsin-like enzyme) are, apparently, true constituents of chromaffin granules.

Nerve growth factor alpha subunit: effect of site-directed mutations on catalytic activity and 7S NGF complex formation.

Mouse alpha- and gamma-nerve growth factor (NGF) are glandular kallikreins that form a non-covalent complex (7S NGF) with beta-NGF. gamma-NGF is an active arginine-specific esteropeptidase; the alpha-subunit is catalytically inactive and has a zymogen-like conformation. Site-directed mutagenesis of alpha-NGF to alter the N-terminus and three residues in loop 7, a region that contributes to the catalytic center, restored substantial catalytic activity against N-benzoyl arginine-p-nitroanilide as substrate in two derivatives although they were not as active as recombinant gamma-NGF. Seven of the 15 derivatives that remained more alpha-like were able to substitute for native alpha-NGF in reforming 7S complexes; the other eight derivatives that were more gamma-like showed greatly reduced ability to do so. However, the most gamma-like alpha-NGF derivative could not substitute for native gamma-NGF in 7S complex formation. These findings suggest that the alpha-NGF backbone can be corrected to a functional enzyme by the addition of a normal N-terminal structure and two catalytic site substitutions and that the 7S complex requires one kallikrein subunit in the zymogen form and one in an active conformation.

The chemokine interleukin-8 regulates parathyroid secretion.

Interleukin-8 (IL-8) is a chemokine important in inflammatory processes. Homology cloning experiments performed using bovine parathyroid cDNA and degenerate primers encoding transmembrane regions III and VI of peptide and protein hormone G-protein coupled receptors identified a set of known receptors not previously identified in the parathyroid. Among these was the IL-8 type B receptor. Incubation of freshly isolated bovine parathyroid cells with recombinant IL-8 for 6-48 h produced an increase in the levels of mRNA for parathyroid hormone (PTH). The levels of PTH secreted in response to nanomolar amounts of IL-8 were also elevated in cells incubated for 1 h with IL-8. Differential display analysis of mRNA from parathyroid cells, incubated in the presence and absence of IL-8, permitted the identification of cDNA clones for RNA species whose expression was either elevated or suppressed. These experiments suggest that IL-8 and inflammatory events play a role in bone homeostasis through actions on the parathyroid gland.

A chromogranin A-derived peptide differentially regulates the secretion of calcitonin gene products.

We studied the regulation of the secretion of CT and CGRP by chromogranin A (CgA)-derived peptides in a human lung tumor cell line. The amino-terminal peptide of CgA, CgA1-40, stimulated the secretion of CGRP and inhibited the secretion of CT; both effects occurred in a dose-dependent manner. These studies demonstrate a differential effect of a CgA-derived peptide on two products of the CT gene, CT itself and CGRP. CgA may be processed at its multiple dibasic sites to peptides that specifically regulate the secretion of its coresident hormones, in this case two calcitonin gene products that are present in the same secretory vesicle. This novel mechanism represents a new pathway for the control of calcium regulating hormones.

Effects of calcium on recombinant bovine chromogranin A.

Bovine chromogranin A, the acidic calcium-binding protein characteristic of endocrine secretory vesicles, has been expressed in Escherichia coli using the pET3a vector system under T7 polymerase control. The expressed protein is located in the bacterial cytosol and can be purified from bacterial proteins by a heat treatment step, followed by gel filtration, anion-exchange, and reversed-phase chromatography. The purified recombinant chromogranin A has an apparent M(r) of ca. 72,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in spite of its 432-amino acid polypeptide chain, consistent with observations on natural chromogranin A. The primary structure has been confirmed by mass spectral analysis of tryptic peptides, by Edman degradation of the intact protein, and by immunoreactivity with sequence-specific antibodies. Analysis by circular dichroism spectroscopy shows pH- and concentration-dependent spectra. The spectra are Ca2(+)-dependent from 5 to 40 microM.

Substrate binding and conformational changes of Clostridium glutamicum diaminopimelate dehydrogenase revealed by hydrogen/deuterium exchange and electrospray mass spectrometry.

C. glutamicum meso-diaminopimelate dehydrogenase is an enzyme of the L-lysine biosynthetic pathway in bacteria. The binding of NADPH and diaminopimelate to the recombinant, overexpressed enzyme has been analyzed using hydrogen/deuterium exchange and electrospray ionization/mass spectrometry. NADPH binding reduces the extent of deuterium exchange, as does the binding of diaminopimelate. Pepsin digestion of the deuterated enzyme and enzyme-substrate complexes coupled with liquid chromatography/mass spectrometry have allowed the identification of eight peptides whose deuterium exchange slows considerably upon the binding of the substrates. These peptides represent regions known or thought to bind NADPH and diaminopimelate. One of these peptides is located at the interdomain hinge region and is proposed to be exchangeable in the "open," catalytically inactive, conformation but nonexchangeable in the "closed," catalytically active conformation formed after NADPH and diaminopimelate binding and domain closure. Furthermore, the dimerization region has been localized by this method, and this study provides an example of detecting protein-protein interface regions using hydrogen/deuterium exchange and electrospray ionization.

Immucillin-H binding to purine nucleoside phosphorylase reduces dynamic solvent exchange.

The rate and extent of hydrogen/deuterium (H/D) exchange into purine nucleoside phosphorylase (PNP) was monitored by electrospray ionization mass spectrometry (ESI-MS) to probe protein conformational and dynamic changes induced by a substrate analogue, products, and a transition state analogue. The genetic deficiency of PNP in humans is associated with severe T-cell immunodeficiency, while B-cell immunity remains functional. Inhibitors of PNP have been proposed for treatment of T-cell leukemia, to suppress the graft-vs.-host response, or to counter type IV autoimmune diseases without destroying humoral immunity. Calf spleen PNP is a homotrimer of polypeptide chains with 284 amino residues, molecular weight 31,541. Immucillin-H inhibits PNP with a Kd of 23 pM when only one of the three catalytic sites is occupied. Deuterium exchange occurs at 167 slow-exchange sites in 2 h when no catalytic site ligands are present. The substrate analogue and product prevented H/D exchange at 10 of the sites. Immucillin-H protected 32 protons from exchange at full saturation. When one of the three subunits of the homotrimer is filled with immucillin-H, and 27 protons are protected from exchange in all three subunits. Deuterium incorporation in peptides from residues 132-152 decreased in all complexes of PNP. The rate and/or extent of deuterium incorporation in peptides from residues 29-49, 50-70, 81-98, and 112-124 decreased only in the complex with the transition state analogue. The peptide-specific H/D exchange demonstrates that (1) the enzyme is most compact in the complex with immucillin-H, and (2) filling a single catalytic site of the trimer reduces H/D exchange in the same peptides in adjacent subunits. The peptides most highly influenced by the inhibitor surround the catalytic site, providing evidence for reduced protein dynamic motion caused by the transition state analogue.

Determination of residues in chromogranin A-(16-40) required for inhibition of parathyroid hormone secretion.

Chromogranin A (CGA), which is cosecreted from the parathyroid gland with PTH in response to low extracellular calcium, can be processed to amino-terminal peptides that, in turn, inhibit PTH secretion. The synthetic peptide KCIVEVISDTLSKPSPMPVSKECFE [CGA-(16-40)] is active in inhibiting secretion from freshly isolated or cultured bovine parathyroid cells. Peptide analogs in which alanine is substituted for classes of residues between the two cysteines have been synthesized and tested for biological activity. Substitution of the lysine, serine, or threonine residues by alanines does not greatly diminish biological activity. However, when the prolines are replaced by alanines or when glutamic acid and aspartic acid are replaced by alanines, the peptides do not effectively inhibit PTH secretion. Tests of synthetic peptides in which the individual glutamate or aspartate residues have been replaced showed that glutamate 37, followed by aspartate 24, are more critical for biological activity. Further experiments have shown that residues 11-15 in the natural CGA sequence do not enhance the biological activity of CGA-(16-40), whereas adding residues 6-10 restores full biological activity compared to that of CGA-(1-40). Circular dichroism experiments with CGA-(16-40) and the alanine substitution analogs show significant differences only for the peptide in which the three prolines are replaced. The inactive peptide with two glutamic acids and one aspartic acid replaced by alanine residues has the same circular dichroism spectrum as some of the peptides that are fully active. The N-terminal CGA sequences may tolerate many changes without alteration of biological activity. However, there are specific amino acid residues that are required for biological function.

Inhibition of parathyroid hormone secretion by amino-terminal chromogranin peptides.

The parathyroid gland responds to decreases in levels of extracellular calcium by increasing the secretion of both PTH and chromogranin-A (CGA) in approximately equal molar ratios. Because CGA has been suggested to be a precursor for biologically active peptides, we used primary cultures of bovine parathyroid cells to examine the effects of various peptides from CGA as well as analogous peptides from chromogranin-B (CGB) on PTH secretion. In concentrations from 10(-8)-10(-7) M, amino-terminal peptide CGA-(1-76) effectively inhibited the release of PTH in response to low extracellular calcium. Truncated analogs of this peptide, CGA-(1-40), CGB-(1-41), and CGA-(17-38) were also found to be active in the following order: CGA-(1-76) = CGA-(1-40) = CGB-(1-41) > CGA-(17-38). The biological activity of CGA-(1-40) was markedly reduced after reduction and alkylation, which resulted in disruption of the single disulfide bond between Cys17 and Cys38. Moreover, peptides derived from other regions of CGA and CGB, which included CGA-(403-428), CGB-(1-16), CGB-(316-326), and CGB-(635-657) were inactive. Pulse-chase experiments, using primary cultures of bovine parathyroid cells, revealed the presence of a CGA peptide in the culture medium that had the same amino-terminal sequence and mobility on sodium dodecyl sulfate-polyacrylamide gels as synthetic CGA-(1-76). Furthermore, in binding and cross-linking studies using intact parathyroid cells, CGA-(1-40) formed a single, covalently linked protein complex with a mol wt of 78,000. Formation of the protein complex could be completely inhibited in the presence of an excess of either CGB-(1-41) or CGA-(17-38). These results show that a naturally occurring amino-terminal peptide from CGA as well as shorter analogs can act as potent inhibitors of PTH secretion, and that their biological activity may be mediated through binding to a specific cell surface protein.

PTHrP secretion is stimulated by CT and inhibited by CgA peptides.

We studied the regulation of the secretion in vitro of the parathyroid hormone-related protein (PTHrP) associated with the hypercalcemia of malignancy by Chromogranin A (CgA)-derived peptides and by human calcitonin (CT) in the BEN human lung tumor cell line. The amino terminal peptide of CgA, CgA1-40, inhibited the secretion of PTHrP, whereas other peptides had no such effect. Human CT stimulated the secretion of PTHrP, whereas other hormones had no such effect. Both effects occurred in a dose-dependent manner. These studies reveal novel regulatory pathways among peptides and proteins that are commonly associated with each other and can have paracrine interactions. CgA may be processed at its multiple dibasic sites to peptides that regulate the secretion of its co-resident hormones, in this case PTHrP. In addition to a paracrine effect, CT may be clinically useful as a provocative agent for PTHrP secretion. Complex interactions are present among the calcium-regulating hormones and their associated proteins.

Adrenal chromaffin granules and secretory granules from thyroid parafollicular cells have several common antigens.

The presence of various antigens in two types of isolated endocrine vesicles (chromaffin granules and secretory vesicles of thyroid parafollicular cells) was investigated by immunoblotting. The two types of vesicles have three common secretory proteins: chromogranin A, chromogranin B and secretogranin II. Furthermore, six common membrane antigens were found: cytochrome b-561, carboxypeptidase H, glycoprotein II, glycoprotein III, synaptin/synaptophysin and SV 2. These results demonstrate that vesicles obtained from neural crest-derived endocrine cells not only share several common secretory peptides and proteins, but also have common properties as far as their membrane antigens are concerned.

Processing of chromogranin A within chromaffin granules starts at C- and N-terminal cleavage sites.

Specific antisera were raised against synthetic peptide fragments of bovine chromogranin A. The soluble proteins of bovine chromaffin granules were subjected to two-dimensional immunoblotting with these antisera. The endogenous breakdown products of chromogranin A gave distinct patterns of immunostaining which enabled us to correlate these peptides with defined regions of the chromogranin A molecule. The results establish that within chromaffin granules degradation of chromogranin A by the endogenous proteases can start either at the C- or the N-terminal site.

Immunological characterization of the endoproteases PC1 and PC2 in adrenal chromaffin granules and in the pituitary gland.

Specific antisera against synthetic fragment of the endoproteases, PC1 and PC2, were used to characterize these proteins. In one-dimensional immunoblots these antisera labelled components of 85 kDa for PC1 and of 70 kDa for PC2 in purified bovine chromaffin granules and anterior and posterior pituitary of ox and rat. In membranes of bovine chromaffin granules glycoprotein H was identified as the major PC2 immunoreactive spot. A major part of these endoproteases appeared membrane bound.

Attraction of human monocytes by the neuropeptide secretoneurin.

Secretoneurin is a newly discovered 33-amino-acid peptide derived from secretogranin II (chromogranin C) that is found in sensory afferent C-fibers. We show here that secretoneurin triggers the selective migration of human monocytes in vitro and in vivo. Combinations of secretoneurin with the sensory neuropeptides, substance P or somatostatin, synergistically stimulate such migration. The attraction of monocytes represents the first established function of secretoneurin as a sensory neuropeptide.

Chromogranin A and B in adenomas of the pituitary. An immunohistochemical study of 42 cases.

Forty-two pituitary adenomas (10 prolactinomas; three ACTH-, nine GH-, two FSH- and two TSH-secreting adenomas; and 16 clinically nonfunctioning null cell adenomas) were investigated immunohistochemically with antibodies against chromogranin A and B as well as ACTH, GH, prolactin (PRL), FSH, LH, TSH, and alpha-HCG antibodies. For the demonstration of chromogranin B, two different antibodies were used--e.g., a polyclonal antihuman antibody and an antiserum against a synthetic peptide (DK-21, chromogranin B 306-326) present in the chromogranin B amino acid sequence. All tumors were positive for both chromogranin B antibodies. Chromogranin A was found in FSH- (two of two) and TSH- (two of two) secreting adenomas; it was also found in a focal distribution in ACTH- (one of three) and GH- (four of nine) secreting adenomas. Thirteen of 16 null cell adenomas contained chromogranin A, whereas no chromogranin A was found in prolactinomas. We conclude that null cell adenomas may arise either from FSH/LH or TSH cells (null cell adenomas with both chromogranin A and B positivity) or from ACTH, GH, or PRL cells (the respective tumors are only positive for chromogranin B). Chromogranin B may be used as a universal marker for pituitary adenomas.

Neuroendocrine cells in intestinal lamina propria. Detection with antibodies to chromogranin A.

This study details immunohistochemical experiments showing the location of chromogranin A-containing cells within the lamina propria of rat and human gut. The presence of this marker confirms the presence of neuroendocrine type cells in the lamina propria.