**2. Structural and biological properties of the antimicrobial peptides derived from chromogranins/secretogranins**

### **2.1 Introduction**

Chromogranins/secretogranins (CGs/SGs) constitute the granin family of genetically distinct acidic proteins present in secretory vesicles of nervous, endocrine and immune cells (Helle, 2004). The natural processing of bovine CGs is well described in granules of sympathoadrenal medullary chromaffin cells, where the resulting peptides are co-secreted with the catecholamines (Metz-Boutigue et al., 1993). The numerous cleavage sites are consistent with the specificity of prohormone convertases (PC1/3 and PC2) and carboxypeptidase E (CPE), that reside within chromaffin granules (Metz-Boutigue et al., 1993; Seidah & Chretien 1999). Secretogranin II (SgII), the third member of the chromogranin family is also processed to generate several natural fragments (Metz-Boutigue et al., 1993; Anouar et al., 1998; Marksteiner et al., 1993; Yajima et al., 2004). The discovery

It was reported that neuropeptides display antimicrobial activities, linking together nervous and immune system (Radek & Gallo, 2007; Sternberg, 2006). Both systems can influence each other: brain and peripheral nervous system directly influence the activity of innate and adaptive immune system. Immune system can relay signals to the nervous system *via* the production of growth factors and cytokines. For example, stress can induce alterations in the immune response (Webster et al. 2002), or can be elicited by infection or injury with release

Exchange between both systems can occur at systemic, as well as at regional or local levels (Sternberg, 2006). The first, global level gathers sympathetic nervous system, the hypothalamic-pituitary-adrenal axis and circulating AMPs. The second, local level, is composed of nervous endings, neuropeptide-releasing cells and receptors-exhibiting cells. At the skin level, important structures, such as Merkel cells (Lucarz & Brand, 2007) localized at the basement membrane, separating epidermis from dermis, are neuropeptide-producing cells, cutaneous nervous cells and target cells. Merkel cells have characteristics of both epidermal and neuroendocrine cells. They are connected to nervous system with terminal sensory synapses and dense-core granules contain CGRP (Calcitonin Gene Related Peptide), VIP (VasoIntestinal Peptide), and CgA (Chromogranin A)-derived peptides (Hartschuh et

Alpha-melanocyte-stimulating hormone (alpha-MSH), a 13 amino-acid peptide, is synthesized by keratinocytes, melanocytes, monocytes and astrocytes (Wikberg et al., 2000). This peptide derives from the pro-opiomelanocortin (POMC) after a processing by a proteolytic cascade (Pritchard & White, 2007), producing also five other peptides. Alpha-MSH acts as an AMP by inhibiting *S. aureus* and *C. albicans* growth at picomolar concentration (Cutuli et al., 2000). Interestingly, the tripeptide KPV (alpha-MSH 11-13) exhibited similar antimicrobial properties (Hiltz & Lipton, 1990; Mandrika et al., 2001; Mugridge et al., 1991), without effect on melanocytes (Sawyer et al. 1990). Alpha-MSH acts in two ways; it has a direct antimicrobial effect at very low concentration and reduces inflammatory responses associated with UV induced epithelial injury (Radek & Gallo, 2007).

**2. Structural and biological properties of the antimicrobial peptides derived** 

Chromogranins/secretogranins (CGs/SGs) constitute the granin family of genetically distinct acidic proteins present in secretory vesicles of nervous, endocrine and immune cells (Helle, 2004). The natural processing of bovine CGs is well described in granules of sympathoadrenal medullary chromaffin cells, where the resulting peptides are co-secreted with the catecholamines (Metz-Boutigue et al., 1993). The numerous cleavage sites are consistent with the specificity of prohormone convertases (PC1/3 and PC2) and carboxypeptidase E (CPE), that reside within chromaffin granules (Metz-Boutigue et al., 1993; Seidah & Chretien 1999). Secretogranin II (SgII), the third member of the chromogranin family is also processed to generate several natural fragments (Metz-Boutigue et al., 1993; Anouar et al., 1998; Marksteiner et al., 1993; Yajima et al., 2004). The discovery

**Neuropeptides in skin immunity** 

of neuropeptides (Brogden et al., 2005).

al., 1989a; Hartschuh et al., 1989b).

**from chromogranins/secretogranins** 

**2.1 Introduction** 

that pancreastatin, a chromogranin A (CGA)-derived peptide inhibits insulin secretion from pancreatic beta-cells, initiated the concept of prohormone (Eiden, 1987; tatemoto et al., 1986). The release of these CGs-derived peptides from chromaffin cells results from the nicotinic cholinergic stimulation and regulates several neuroendocrine functions (Helle & Serck-Hanssen, 1975).

Numerous cleavage products of the granins have been characterized, among which some display biological activities (Tatemoto et al., 1986; Aardal et al., 1993; Curry et al., 1992; Fasciotto et al., 1993; Lugardon et al., 2001; Mahata et al., 1997; Strub et al., 1996a,b). Neuroendocrine activities are reported from *in vivo* studies, with modulations of homeostatic processes, such as calcium regulation and glucose metabolism (Helle et al., 2007), cardiovascular functions ( Brekke et al., 2002; Corti et al., 2004), gastrointestinal motility (Amato et al., 2005; Ghia et al., 2004a), nociception ( Ghia et al., 2004b) tissue repair (Gasparri et al., 1997; Ratti et al., 2000), inflammatory responses (Ceconi et al., 2002; Corti et al., 2000) and as host defense agents during infections (Radek et al., 2008). During the past decade, our laboratory has characterized new antimicrobial CGs-derived peptides (Strub et al., 1996a,b; Metz-Boutigue et al., 1998; Lugardon et al., 2000, 2001; Briolat et al., 2005; Helle et al., 2007) (Figure 3).


Fig. 3. The antimicrobial bovine CGs-derived peptides according to the sequence of CGA (P05059), CGB (P23389) and CGC ((P20616) For each antimicrobial peptide the sequence, the location and net charge are indicated. \*, cysteine residues of the disulfide bridge; phosphorylated residue are underlined and the glycosylated residue is in bold.

They act at micromolar range against bacteria, fungi, yeasts and are non-toxic for mammalian cells. They are recovered in biological fluids involved in defense mechanisms (serum, saliva) and in secretions of stimulated human neutrophils (Briolat et al., 2005; Lugardon et al., 2000).

The Natural Antimicrobial Chromogranins/Secretogranins-Derived

bovine and human VS-I sequences (L42-L49; L57-L64).

biological activity in mammalian tissue (Blois et al., 2006).

produced during infections by this class of pathogens.

dimerization motif helix-helix L57-L64 (Lugardon et al., 2001).

**Chromofungin** 

**Catestatin** 

Peptides – Production, Lytic Activity and Processing by Bacterial Proteases 191

present in DAT and dopamine transporter sequences (Torres et al., 2003) are present in the

Surface interaction of rhodamine-labelled bCGA1-40 was demonstrated using confocal microscopy after incubation of the labeled peptide with *Aspergillus fumigatus*, *Alternaria brassicola* and *Neurospora crassa* (Blois et al., 2006). In addition, the interaction of bCGA1-40 with monolayers of phospholipids and sterols, as models for the interaction with mammalian and fungal membranes was investigated by the surface tension technique (Blois et al., 2006; Maget-Dana et al., 1999). These studies demonstrated that the N-terminal bCGA1-40 fragment interacts with model membrane phospholipids in a manner consistent with an amphiphilic penetration into membranes in a concentration range relevant for

When VS-I was treated with the endoprotease Glu-C from *Staphylococcus aureus*, one of the generated peptide, chromofungin (CHR), is the shortest active VS-I-derived peptide with antimicrobial activities (Figure 3). It is well conserved during evolution and displays antifungal activity at 2-15 µM against filamentous fungi (*Neurospora crassa, Aspergillus fumigatus, Alternaria brassicola, Nectria haematococca, Fusarium culmorum, Fusarium oxysporum*) and yeast cells (*Candida albicans, Candida tropicalis, Candida neoformans*) (Lugardon et al., 2001). Since this peptide was generated after digestion of the material present in chromaffin secretory vesicles by the endoprotease Glu-C from *S. aureus*, it may be hypothesized that it is

The 3-D structure of CHR has been determined in water-trifluoroethanol (50:50) by using 1H-NMR spectroscopy. This analysis revealed the amphipathic helical structure of the sequence 53-56, whereas the segment 48-52 confers hydrophobic character (Lugardon et al., 2001).The importance of the amphipathic sequence for antifungal activity was demonstrated by the loss of such activity against *N. crassa* when two proline residues were substituted for L61 and L64, disrupting the helical structure, the amphipathic character and the

Two CGA-derived fragments bCGA333-364 and bCGA343-362 were characterized after the extensive processing of bCGA by prohormone convertases (PC 1/3 or 2) in chromaffin granules (Taylor et al., 2000). More recently, it was shown that cathepsin L colocalizes with CGA in chromaffin granules*. In vitro* it is able to generate after digestion of recombinant hCGA, a catestatin (CAT)–derived fragment hCGA360-373 (Biswas et al., 2009). In addition to the inhibitory effect of CAT on catecholamine release from chromaffin cells (Mahata et al., 1997), we have shown for this peptide and its shorter active sequence bCGA344-358 (cateslytin, CTL), (Figure 3) a potent antimicrobial activity with a MIC in the lowmicromolar range against Gram-positive bacteria (*Micrococcus luteus, Bacillus megaterium* at concentration of 0.8 µM), Gram-negative bacteria (*Escherichia coli D22* at concentration of 8 µM), filamentous fungi (*Neurospora crassa, Aspergillus fumigatus, Nectria haematococca* at concentration of 0.2-10 µM) and yeasts (*Candida albicans, Candida tropicalis, Candida glabrata, Candida neoformans* at concentration of 1.2-8 µM). The sequence of CAT (Figure 3) has been highly conserved during evolution (Briolat et al., 2005). The two human variants P370L and

These new AMPs are integrated in the concept that highlights the key role of the adrenal medulla in the immunity (Sternberg, 2006) as previously reported for adrenaline and neuropeptide Y that regulate immunity systemically once released from the adrenal medulla. Furthermore, the adrenal medulla contains and releases large amounts of IL-6 and TNF-alpha in response to pro-inflammatory stimuli such as LPS, IL-1 alpha and IL-1 beta (Metz-Boutigue et al., 1998). The discovery of the presence of TLRs on the adrenal cortex cells raises the interesting possibility that the adrenal gland might have a direct role in the response to pathogens, activation of innate immune response and clearing of infectious agents (Sternberg, 2006).
