Presence and release of the chromogranin B-derived secretolytin-like peptide KR-11 from the porcine spleen

Regulatory Peptides 122 (2004) 99 – 107 www.elsevier.com/locate/regpep

Presence and release of the chromogranin B-derived secretolytin-like peptide KR-11 from the porcine spleen Jan Depreitere a, Zesheng Wang a, Fei Liang b, Edmond Coen a, Etienne J. Nouwen a,* a

Laboratory of Neurobiology and Neuropharmacology, Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium b Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Received 12 January 2004; received in revised form 10 May 2004; accepted 1 June 2004 Available online 17 July 2004

Abstract Chromogranin B (CgB) is a major matrix protein in secretory granules/large dense-cored vesicles and a precursor for smaller peptides. In an earlier study, we have identified a secretolytin-like peptide (KR-11, pCgB637 – 647) from porcine chromaffin granules. Further evidence is presented here to show the processing of chromogranin B to this peptide during axonal transport in the splenic nerve and its release in the spleen upon various conditions of stimulation. Immunohistochemical staining showed that in the porcine spleen chromogranin B and NPY completely colocalize in nerve fibres and varicosities in blood vessel walls and trabeculae, and along the loose network of smooth muscle cells in the red pulp, as identified by their a-smooth muscle cell actin content. No antibacterial activity was found for the porcine secretolytinlike peptide, KR-11. The change of one amino acid residue (Thr ! Asn) in the porcine homologous fragment of secretolytin appears to be responsible for its loss of antibacterial activity. D 2004 Elsevier B.V. All rights reserved. Keywords: Secretogranin I; Processing; Sympathetic nerve; Function

1. Introduction Chromogranin B (CgB)/secretogranin I is an acidic protein belonging to the chromogranin family which comprises chromogranin A (CgA) and B [1], secretogranin II (SgII) [2], 7B2 [3] and NESP55 [4]. All these proteins are found in secretory granules/large dense-cored vesicles (LDCV) throughout the endocrine and nervous systems [1– 4]. They are co-stored and secreted together with resident hormones and transmitters via a regulated pathway [5,6]. CgB has frequently been used to study sorting and trafficking in the regulated pathway of protein secretion [7]. It has been reported that CgB binds to the secretory vesicle membrane [8] by interaction with an intraluminal loop peptide of the inositol 1,4,5-triphosphate receptor on that membrane [9]. Evidence suggests that CgB may have multiple roles in secretion. On the one hand, intracellular CgB can act as a helper protein in the packaging of peptide hormones and neurotransmitters [10 –12], and on the other * Corresponding author. Tel.: +32-3-820-25-69; fax: +32-3-820-25-67. E-mail address: [email protected] (E.J. Nouwen). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.06.002

hand, extracellular CgB regulates hormone secretion [13]. A functional study demonstrated that CgB regulates islet amyloid polypeptide (IAPP) and insulin secretion [13]. In two regions of CgB, there is a high degree of homology between the individual mammalian species [14,15]. One is located near the N-terminus and the other at the actual Cterminus [15], implying important roles for these two regions. CgB has been shown to undergo proteolytic processing at both the N- and C-terminus. Because of the occurrence of numerous CgA- and CgB-derived peptides, an extracellular role as a precursor of biologically active peptides has already been proposed [1,2,16]. Secretolytin, a new antibacterial peptide corresponding to the 614– 626 C-terminal region of bovine CgB, has been discovered by Strub et al. [17,18]. Recently increased blood levels of the human homologue of secretolytin were found during surgery in patients undergoing cardiopulmonary bypass, where it is released from monocytes and reported to possess antibacterial activity [19]. A similar CgB-derived fragment has also been identified from porcine chromaffin granules in our laboratory, namely KR-11 (pCgB637 – 647). Sequencing and molecular mass analysis revealed that this

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peptide is homologous to bovine secretolytin but it lacks the N- and C-terminal amino acid [20]. Our previous results suggested that KR-11 is produced inside the chromaffin granules and not in the extracellular compartment. In the present study, immunohistochemical staining experiments were performed to show and compare the distribution of CgB- and NPY-containing nerve fibres in the porcine spleen. Furthermore, the proteolytic processing and co-release of KR-11 together with noradrenaline (NA) and neuropeptide Y (NPY) was demonstrated in the porcine splenic nerve under various conditions of stimulation. In addition, sequence comparison of the porcine secretolytin homologous fragments with bovine secretolytin reveals that the change of one amino acid residue appears to be responsible to its loss of antibacterial activity.

2. Materials and methods 2.1. Immunohistochemistry The following primary antibodies were used: a rabbit antiserum against synthetic SR-17 (pCgB586 – 602), custommade by CER, Laboratory of Hormonology, Marloie, Belgium (dilution: 1:1000), a rabbit anti-porcine NPY antiserum (Sanver Tech, Belgium) (dilution: 1:1000) and a mouse monoclonal antibody against porcine a-smooth muscle actin (clone IgG2a A2547; Sigma) (dilution: 1:100,000). Staining for CgB was performed essentially as described previously [21]. Briefly, 2-mm thick slices of pig spleen, obtained from the slaughterhouse, were immersion-fixed in formol – calcium fixative (4% formaldehyde in 0.121 M Na – cacodylate, pH 7.4, containing 1% CaCl2) for 90 min at room temperature, washed with Na – cacodylate buffer (0.121 M containing 1% CaCl2) and embedded in paraffin. Five-micrometer sections were mounted on poly-L-lysinecoated glass slides and rehydrated. After washing with Tris – saline buffer (TSB: 0.01 M Tris – HCl, pH 7.6, containing 0.9% NaCl and 0.1% Triton X-100) and treatment with 20% normal goat serum for 20 min, the primary antibody was applied without washing, and incubated overnight. Endogenous peroxidase was blocked with 0.03% H2O2. Further processing was performed according to an avidin biotin – peroxidase complex method (Vector Laboratories, Burlingame, CA, USA). Biotinylated goat-anti-rabbit IgG or horse-anti-mouse IgG was applied for 30 min, followed by a 1-h incubation with the avidin –biotin – horseradish peroxidase complex. All dilutions were made in TSB, containing 0.1% BSA. Peroxidase enzyme activity was revealed using 3-amino-9-ethylcarbazole (AEC) in 20 mM NaAc buffer, pH 5.2, containing 9.5% DMSO and 0.002% H2O2. Sections were counterstained with methylgreen and mounted in Kaiser’s glycerin/gelatin mounting medium. For an optimal comparison of the staining patterns for CgB and NPY, two AEC immunoperoxidase staining sessions were applied consecutively on the same sections. After

the first staining (for CgB), photomicrographs were taken of selected areas. Then the AEC reaction product was removed by immersion in isopropanol, and the sections were incubated for 20 min at 98 jC in 0.01 M Na –citrate buffer, pH 6.0, to remove all immunoreagents. After cooling down, they were stained for NPY and counterstained. Photographs were taken at the same positions as previously for CgB. Control sections, which underwent the whole procedure without the primary antibody for NPY, showed no signal upon the second staining, thereby demonstrating that all immunoreagents from the first procedure had been successfully removed. For double staining of CgB and a-smooth muscle cell actin, sections were incubated at 98 jC in 0.01 M Na – citrate, pH 7.4, for 20 min after the AEC staining for CgB, to remove the first primary and secondary antibody and ABC complex. The sections were then treated for 20 min with 0.02% trypsin in 0.01 M Tris – HCl, pH 7.3, containing 0.9% NaCl and 0.001 M CaCl2. After washing with TSB, they were blocked again with 20% normal goat serum and the primary antibody against a-smooth muscle actin was added and sections were incubated overnight at 4jC, followed by the second ABC procedure in which alkaline phosphatase was used as an enzyme marker. Alkaline phosphatase enzyme activity was demonstrated with NBT [21], which produces a blue staining. L-p-Bromotetramizole was added as an inhibitor of endogenous alkaline phosphatase activity. Sections were counterstained and mounted as explained above. Staining patterns were analysed using an Olympus light microscope (type BX40) and digital photomicrographs were taken using a Sony 3 CCD colour video camera (Type DXC-950). 2.2. Tissue extraction Porcine adrenal medulla (n = 70), splenic nerve (n = 15) and spleen (n = 1) were obtained from a local slaughterhouse. The tissues were transferred to ice-cold buffer (0.3 M sucrose, 5 mM Tris, 1 mM MgCl2, pH 7.3) and were dissected, weighed and homogenised for 30 s in 10 ml of distilled water/g wet weight at 4 jC with an Ultra-Turrax homogeniser. The homogenates were sonicated for 10 s, boiled for 10 min and centrifuged at 14,000  g for 20 min (4 jC). The supernatants were lyophilized and analysed by gel filtration chromatography followed by ELISA. Three experiments were carried out for each tissue. 2.3. Perfusion The experiments were carried out as described earlier [22,23] with some minor modifications. In brief, the spleen was perfused with aerated (95% O2 – 5% CO2) Krebs – Ringer bicarbonate solution (containing 2000 I.U./kg heparin during the first 30 min) at a flow rate of 10 ml/min for about 90 min, with initially three injections of adrenaline

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(330 Ag/ml/injection) to contract the spleen in order to remove excess of blood. Subsequently, the spleen was perfused for another 40 min before collecting samples. Perfusates were collected during a 5-min period before, during and after electrical stimulation in ice-chilled polypropylene tubes containing sodium – EDTA (10 mM) and phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich, Bornem, Belgium) (1 Ag/ml). Each experiment consists of six consecutive stimulations: twice at 2 Hz in the absence of phentolamine, followed by twice at 2 Hz in the presence of phentolamine (5 AM) and, finally, twice at 5 Hz in the presence of phentolamine. In some experiments, 10 Hz was applied in the presence of phentolamine. The average value of two consecutive stimulations was calculated. The interval between consecutive stimulations was 30 min. Phentolamine was infused after the second stimulation (i.e., 30 min before the third stimulation) and lasted until the end of the experiment. Perfusate samples (50 ml) were immediately centrifuged (3000  g, 15 min, 4 jC) to remove blood cells, and subsequently aliquots were taken for measurement of noradrenaline (NA) and proteins. The remaining sample was boiled for 12 min, cooled on ice, and centrifuged for 45 min at 110,000  g (4 jC). The peptides were extracted by SepPak C18 solid-phase columns (Waters, Ireland) and concentrated by lyophilisation (Speedvac, Savant, Farmingdale, NY, USA). The lyophilised perfusates were analysed by the KR-11 ELISA and the NPY-RIA.

The antiserum was coupled to CNBr-activated Sepharose 4B beads (Pharmacia Biotech, Sweden). In short, before coupling, the antibody was precipitated with saturated (NH4)2SO4 and dialysed against the coupling buffer (0.1 N NaCO3, pH 8.3, 0.5 M NaCl). The required amount of sepharose beads was weighed, swollen for 15 min in 1 mM HCl and washed with 1 mM HCl and coupling buffer. The coupling solution containing the antibody was mixed with the gel in a rotating vessel at 4 jC overnight (2 mg antibody/ml beads). The excess of ligand was washed away with at least five gel volumes of coupling buffer. The remaining groups were blocked by bringing the gel in 0.1 M Tris – HCl buffer, pH 8.0, for 2 h. Afterwards, the column is filled with the gel and washed with at least three cycles of alternating pH. Each cycle consists of a wash with 0.1 M acetate buffer, pH 4.0, containing 0.5 M NaCl, followed by a wash with 0.1 M Tris – HCl buffer, pH 8.0, containing 0.5 M NaCl. The peptides extracted from spleen homogenates with Sep-Pak C18 (Waters, Milford, MA, USA) were adsorbed to the secretolytin affinity column by slow passage at 4 jC. The column was washed with 20 column volumes of PBS buffer, pH 7.4. The peptides were eluted with one column volume of 0.1 M glycine, pH 2.4, and one volume of 10% dioxane in Tris – HCl buffer, pH 8.3.

2.4. Gel filtration chromatography Lyophilised aliquots from different tissue extracts and perfusates were resuspended in the eluent buffer. Samples were then loaded on a Superose 12 HR 10/30 gel filtration column (Pharmacia LKB, Sweden) equilibrated with 75 mM NaH2PO4, pH 7.4, containing 75 mM NaCl and 30% acetonitrile (eluent buffer), and eluted at a flow rate of 0.4 ml/min on a Fast Protein Liquid Chromatography (FPLC) system. The eluent was monitored by absorbance at 214 nm. One-minute fractions were collected.

Reversed-phase chromatography was performed on a Waters HPLC system equipped with a PDA detector. The concentrated eluents from the immunoaffinity and gel filtration columns were applied to a YMC C18 column (ODS˚ , 4.6  250 mm). The solvent system AP, S-5 Am, 300 A consisted of 0.1% trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic acid in acetonitrile (solvent B). Elution was performed at a flow rate of 1 ml/min, by a threestep gradient: 0 – 20% (10 min), 20 –50% (30 min) and 50– 90% (10 min). Fractions were collected, concentrated by lyophilisation and analysed by ELISA.

2.5. Immunoaffinity chromatography

2.7. KR-11 ELISA

A specific antiserum against KR-11 was custom-made (Eurogentec, Lie`ge, Belgium) by immunizing rabbits with synthetic KR-11, coupled to keyhole limpet hemocyanin using the MBS (m-Maleimidobenzoyl-N-hydroxy-succinimide ester) reagent, which makes a link between a cystein placed at the N-terminus of KR-11 and NH2 groups of the carrier protein. The antibody recognizes the immunoreactive epitope not only in the free peptide, but also in the large precursor. The specificity of this antiserum was tested by preincubation with 100 Ag of CgA1 – 13, CgB1 – 17 and SgII66 – 83. The immunoblotting pattern showed that this antiserum does not recognize the N-terminal fragment of CgB and that it does not cross-react with the peptides derived from CgA or SgII.

Ninety-six-well plates (Costar, Corning, NY, USA) were coated with 2 ng of synthetic KR-11 in 100 Al coating buffer (10 mM Tris, 10 mM NaCl and 10 mM sodium azide, pH 8.6) at 37 jC for 3 h. After blocking, the plates with 200 Al blocking buffer (PBS + 0.1% caseine) for 1 h at 37 jC, standard KR-11 (from 2560 pg to 20 pg/100 Al) or sample were added to the wells (in triplicate), followed by 50 Al of the rabbit anti-KR-11 antiserum (1:6000). Plates were incubated overnight at 4 jC. After five washings with wash solution (PBS + 0.05% Tween 20, pH 7.4), the wells were incubated with biotinylated, affinity-purified anti-rabbit IgG for 30 min at 37 jC, followed by an incubation in avidin– biotin – peroxidase complex (Vectastain ABC system; Vector Laboratories, Burlingham, CA, USA) for 30 min at 37 jC.

2.6. Reversed-phase HPLC

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Finally 100 Al of the substrate solution (3,3V,5,5Vtetramethylbenzidine (TMB) in sodium acetate + 0.01% hydrogen peroxide) was added to the wells. After a 30min incubation in the dark at room temperature, 50 Al of 2 M H2SO4 was added to stop the reaction and absorption of each well was measured at 450 nm with a verticallightpath microplate reader in the dual-wavelength mode (filter 1, at 450 nm; filter 2, at 690 nm; Titertek Multiskan MCC/340 MK II; Flow Laboratories, Helsinki, Finland). The ELISA results were calculated by a four parameter logistic function by means of an ELISA data reduction program. The specificity of the antiserum against KR-11 was described above. The standard curve ranged from 5 pg to 5 ng/100 Al. Correlation coefficients of 0.99 or more were obtained. The intra-assay coefficient of variation, calculated from quadruplicate measurement of chromaffin granule lysate, was 4%.

2.11. Antibacterial activity Antibacterial activity was analysed using the microbroth dilution method [26]. Test peptides were dissolved in the broth at 1.0 mg/ml, bovine chromaffin lysate at 1.5 mg/ml, and porcine chromaffin lysate at 1.0 mg/ml. Test proteins or peptides in media were diluted twofold in serial doubling dilutions and the microbiological inoculum was added to each well, and incubated at 37 jC overnight. Bacterial growth was detected by measuring the OD600 nm. The following microorganisms were used: Bacillus cereus, Escherichia coli, Micrococcus luteus, Pseudomonas aeruginosa, Salmonella thyphimurium and Staphylococcus aureus.

3. Results

2.8. NPY radioimmunoassay

3.1. Immunohistochemical distribution and colocalisation of CgB and NPY in the porcine spleen

RIA was performed using a commercial NPY-RIA kit (Euro-Diagnostica, Sweden). In brief, NPY in the samples is assayed by a competitive radioimmunoassay using an antiserum raised against synthetic human NPY. This antiserum cross-reacts 100% with porcine NPY, and less than 2% and 1% with human peptide YY and pancreatic polypeptide. NPY was used as a tracer (125I-labelled) and as a standard. The antiserum (final dilution 1:16,000) was incubated with standards and samples for 24 h at 4jC in RIA buffer (0.05 M phosphate buffer, pH 7.4, with 0.25% human serum albumin, 0.25% EDTA, 0.1% Triton X-100, 0.05% NaN3, and 500 KIU TrasylolR per ml). The tracer (104 cpm) was added to the standards and the samples, which were then incubated for an additional 24 h at 4 jC. Bound and free fractions were separated by adding 0.1 ml of doubleantibody solid phase (anti-rabbit-Ig coupled to cellulose particles). After a 30-min incubation at 4 jC, samples were centrifuged at 3200  g for 15 min at 4 jC. The radioactivity of the antibody-bound 125I-NPY was measured in a g-counter. The detection limit is 6 pmol/l and a mean recovery of 85% was achieved.

Sections consecutively stained with antisera against porcine CgB and NPY showed virtually identical staining patterns. The trabeculae and the loose network of isolated smooth muscle cells/reticular cells throughout the red pulp (Fig. 1A,B), which were identified as such by their asmooth muscle actin positivity (Fig. 2A,B), were innervated by nerve fibres displaying numerous both CgB and NPYcontaining varicosities. Larger branches of the splenic nerve were also positive for both substances (Fig. 1C,D). In the white pulp, staining was confined to positive nerve fibres forming plexuses surrounding the central arterioles and their branches (Fig. 3). A similar staining pattern was seen in blood vessel walls—arterioles, veins and venules—in the red pulp. Some positive nerve fibres were also found in the splenic capsule. Thus, an extensive association of CgB- and NPY-containing nerve fibres with smooth muscle cells was seen in trabeculae, the diffuse network in the red pulp, blood vessel walls, and capsule. In contrast, splenocytes were always negative for CgB and for NPY.

2.9. Catecholamine and protein determination

3.2. Proteolytic processing of CgB during axonal transport

NA was determined by high-performance liquid chromatography with electrochemical detection (HPLC-ED) as described before [24]. Protein concentrations were measured with Coomassie brilliant blue G-250 according to Bradford [25], using bovine serum albumin as a standard.

To study the generation of KR-11 in sympathetic axons and nerve terminals, boiled extracts of porcine splenic nerve (Fig. 4B), spleen (Fig. 4C) and spleen perfusate (Fig. 4D) were size-fractionated on a Superose-12 column and analysed for KR-11 immunoreactivity (IR) by ELISA. For comparative reasons, this analysis was also performed on adrenal medulla (Fig. 4A). In all cases, the chromatographic profile revealed a peak at 43 min, which corresponds to the retention time of synthetic pKR-11. In the adrenal medulla, the endoproteolytic processing of CgB is rather limited. The intact

2.10. Peptide synthesis Peptides KR-11 (pCgB637 – 647) and QG-13 (pCgB636 – 348) were synthesized by Eurogentec and were purified by RP-HPLC.

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Fig. 1. Consecutive immunoperoxidase staining, on the same section, for CgB (A and C) and NPY (B and D) in porcine spleen. The distribution pattern of the two neuropeptides is identical. Double-positive varicosities are abundant in trabeculae (arrowheads) and along isolated smooth muscle cells/reticular cells throughout the red pulp (arrows). Branches from the splenic nerve (asterisk) were also positive for both substances (C and D). Bar = 40 Am.

CgB precursor and intermediate-sized peptides were the major molecular forms present in the adrenal medulla (7% and 42% of the total IR, respectively). In contrast, in the splenic nerve the intermediate and the smaller peptides predominated (18% and 46%, respectively), whereas the CgB precursor protein is absent. Apparently, in the nerve terminals of the spleen, smaller peptides are dominant or represent at least a significant proportion of the total immunoreactivity (43% for the smaller peptides and 10% for KR-11). The highest total (i.e., processed and

Fig. 2. Combined immunostaining for CgB (red staining; arrowheads) and a-smooth muscle actin (blue staining; arrows). CgB-containing nerve fibres are always associated with smooth muscle cells in trabeculae (A) and the diffuse network in the red pulp (B). Bar = 20 Am.

Fig. 3. Immunoperoxidase staining for CgB. Positive varicosities (arrows) in the wall of central arteries in the periarteriolar lymphoid sheaths (PALS). Bar = 40 Am.

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at two different frequencies were carried out in the presence and absence of the a-blocker phentolamine. In all experiments, two successive stimulations with the same frequency (2 Hz, 5 min) were followed by two successive stimulations with another frequency (5 Hz, 2 min), but applying the same total number of pulses (600). The perfusates were analysed for KR-11 by ELISA, for NA by HPLC-ED and for NPY by RIA. The release of NA, NPY and KR-11 increased with increasing frequencies (Fig. 5). Furthermore, in the presence of phentolamine, the release of NA, NPY and KR-11 upon stimulation at 2 Hz for 5 min markedly increased (about 2.5 – 4 times) as compared to the control stimulation (2 Hz for 5 min, without phentolamine). The release could further be increased by increasing the frequency of stimulation to 5 Hz (Fig. 5). 3.4. Demonstration of the presence of KR-11 free peptide in spleen and spleen perfusate after stimulation Affinity-purified porcine spleen extract and stimulated spleen perfusate (10 Hz, 1 min) were both subjected to FPLC-gel filtration. Fraction 43, corresponding with the elution position of synthetic KR-11, from both the spleen extract and the perfusate was further subjected to reversed-phase HPLC. The fractions obtained were analysed

Fig. 4. FPLC-gel filtration chromatography of porcine sympathetic neuronal tissue extracts. Boiled extracts of adrenal medulla, splenic nerve, spleen and spleen perfusate were applied to a gel filtration column. Fractions were analysed for KR-11 IR by ELISA. The elution positions of the precursor CgB and of the free secretolytin-like peptide, KR-11, are indicated by arrows.

unprocessed) tissue levels were found in the adrenal medulla, whereas the highest relative abundance of the free peptide was seen in the spleen. In the stimulated spleen perfusate (Fig. 4D), there is only one single peak present, eluting at 42 – 44 min. KR-11 represents 19% of the total IR. No KR-11 IR could be detected in any samples examined at the position of intact CgB (position 25). 3.3. Release of KR-11 IR In order to confirm the co-release of KR-11, NA and NPY, an established co-transmitter from sympathetic neurons, consecutive electric stimulations of the splenic nerve

Fig. 5. Effect of splenic nerve stimulation on the differential release of NA, NPY and KR-11 IR from the spleen, at low frequency (2 Hz), in the presence and absence of phentolamine, and high frequency (5 Hz) in the presence of phentolamine, giving the same total number of pulses (600). Each value represents the mean F S.E.M. from three perfusions (C = control; 2N = 2 Hz in the absence of phentolamine; 2P = 2 Hz in the presence of phentolamine; 5P = 5 Hz in the presence of phentolamine).

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Fig. 6. Representative reversed-phase HPLC profile of KR-11 IR in gel filtration fraction 43 of affinity-purified spleen extracts and spleen perfusate after stimulation (10 Hz, 1 min). KR-11 IR co-eluted with the synthetic peptide standard at 16 min.

for KR-11 by ELISA. The chromatographic elution profile revealed one major peak eluting in both cases exactly at the position of the secretolytin standard (Fig. 6), thereby demonstrating the presence of the free peptide in sympathetic nerve terminals and its release from the stimulated spleen. 3.5. Antibacterial activity of KR-11 Bovine and porcine chromaffin granule lysates and synthetic KR-11 were tested against several Gram-positive and -negative bacteria. The chromaffin lysates displayed antibacterial activity against M. luteus and B. cereus at a minimal inhibitory concentration of 375 and 250 Ag/ml, respectively, but no activity was found for pKR-11 for concentrations up to 1 mg/ml. Since the peptide length has been reported to be related to its activity [17], QG-13 (pCgB636 – 648) was synthesized having the same length as bovine secretolytin but with the sequence of porcine CgB, including the two extra amino acid residues (Table 1), and was tested for its antibacterial activity. Again, no activity could be detected. Table 1 Secretolytin sequence homology between different species and its antibacterial activity Name

Sequence

Antibacterial activity

Reference

Porcine KR-11 Porcine QG-13 Bovine secretolytin Human homologous fragment

– KIAE KFSGN RR – QKIAE KFSGN RRG QKIAE KFSGT RRG QKIAE KFS – – QRG

No No Yes Yes

This study This study [18] [19]

4. Discussion In the present study, we show that all CgB-containing nerve fibres in the porcine spleen overlap with those containing NPY. Moreover, we show the presence of CgB in tissue extracts, its processing to the mature peptide KR-11 within sympathetic neurons, and its release from the porcine spleen upon nerve stimulation. A significant processing of CgB has been shown in the rat [27] and human brain [28]. Much less, however, is known about its processing in peripheral nerves. We studied the processing of CgB and the production of KR-11 in the porcine splenic nerve during axonal transport by gel filtration chromatography followed by ELISA. Depending on the tissue, CgB is proteolytically processed to various degrees, i.e., from partial processing in the adrenal medulla to total processing in nerve terminals (spleen perfusate). During axonal transport, the intermediately sized molecules were further processed to the free peptide. When the LDCV have reached the nerve terminal, the processing is almost complete. This is consistent with our previous study concerning the porcine CgB-derived peptide, SR-17 [29]. The frequency-dependent release of NA and NPY by exocytosis at nerve terminals is well-established [30]. Our observation that the release of KR-11 into the spleen perfusate follows this pattern, adds further support to the conclusion that this peptide is released from the LDCV. In the presence of an a-blocking agent (phentolamine), a drug especially chosen to cause a dramatic increase in the release of substances from LDCV by inhibiting the negative feedback system of the transmitter NA, NA, NPY and KR-11 are still released in the same proportion. This parallel change in release of NA, NPY and KR-11 provides conclusive evi-

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dence that release of KR-11 occurs from sympathetic neurons and, more specifically, from LDCV. In our previous study using immunofluorescence, we found that secretoneurin, a functional peptide derived from SgII, was present in a very low density in the trabeculae and around the vessels of porcine spleen [31]. In this study, we showed the immunohistochemical distribution of CgB and compared it with that of the wellknown co-transmitter NPY, the distribution of which has been extensively documented in the rat [32], beluga whale [33] and porcine spleen [34]. Using light microscopy, fibres were observed along the capsula, trabeculae, venous systems, and along the arterial systems including prominent innervation of the central arterioles in the white pulp. A striking difference was obtained between the red and white pulp with regard to the occurrence of CgB positive nerves, which were associated mainly with red pulp. Although it cannot be excluded that some of the CgBpositive nerve terminals may make contact with leukocytes, at least the great majority of them are in close association with smooth muscle cells in blood vessel walls, trabeculae and the loose arrangement of a-smooth muscle actin-positive cells throughout the red pulp. CgBimmunoreactive profiles completely overlapped with those for NPY. The C-terminal region of chromogranin B is wellconserved during evolution, suggesting an important role. Although an antibacterial activity has been described for bovine secretolytin (1 –2 AM) [18] and for the human homologous fragment [19], no such activity could be observed in the present study for porcine secretolytin-like peptide, KR-11, and for its longer form QG-13, even at 620 AM. Sequence comparison (Table 1) shows that only one amino acid residue (threonine, Thr) in bovine secretolytin is changed to asparagine (Asn) in the porcine QG13 homologous fragment. The human homologous fragment shows the deletion of two amino acid residues at this site. The predicted secondary structure by GOR3 [35] for the bovine, human and porcine homologous fragments of secretolytin show some differences. The N-terminal region (amino acid residues 1 –7) of both bovine secretolytin and the porcine homologous fragment forms a single a-helical segment. The C-terminal region of bovine secretolytin tends to an extended strand, while that of the porcine homologous fragment tends to a random coil due to the substitution of residue Thr ! Asn. The almost entire human homologous secretolytin (nine amino acids) has the tendency to form a a-helix and the secondary structure of bovine secretolytin has been experimentally confirmed as a-helix [17]. An a-helical amphipathic structure for the whole molecule has been found to be necessary for the antibacterial activity of secretolytin [17,18]. Therefore, the change of one amino acid residue (Thr ! Asn) in porcine secretolytin homologous fragment QG-13 has an important effect on its secondary structure and, consequently, on its function.

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