Structure and antimicrobial activity relationship of royalisin, an antimicrobial peptide from royal jelly of Apis mellifera.

Royalisin is a 5.5-kDa antibacterial peptide isolated from the royal jelly of the honeybee (Apis mellifera). The antimicrobial activity of royalisin against fungi, Gram-positive and Gram-negative bacteria has been revealed. Compared with another insect antibacterial peptide, there is an extra stretch of 11 amino acid residues at the C-terminus of royalisin. In this study, a recombinant shortened form of royalisin named as royalisin-D, was constructed without the 11 amino acid residues at the C-terminal of royalisin and linked to the C-terminal of oleosin by an inteinS fragment. The recombinant protein was overexpressed in Escherichia coli, purified by artificial oil body system and subsequently released through self-splicing of inteinS induced by the changes of temperature. The antibacterial activity of royalisin-D was compared with royalisin via minimal inhibitory concentration (MIC) assay, minimal bactericidal concentration (MBC) assay, microbial adhesion to solvents (MATS) methods, and cell membrane permeability. Furthermore, the recombinant royalisin and royalisin-D have also been treated with the reducing agent of disulfide bonds, dithiothreitol (DTT), to investigate the importance of the intra-disulfide bond in royalisin. In our results, royalisin-D exhibited similar antimicrobial activity to royalisin. Royalisin and royalisin D lost their antimicrobial activities when the intra-disulfide bonds were reduced by DDT. The intra-disulfide bond plays a more important role than the extra stretch of 11 amino acid residues at the C-terminus of royalisin in terms of the antimicrobial properties of the native royalisin.

New criterion for evaluation of honey: quantification of royal jelly protein apalbumin 1 in honey by ELISA.

The 55 kDa major protein of royal jelly, named apalbumin 1, is an authentic protein of honey and pollen pellet, and for its quantification an enzyme-linked immunosorbent assay (ELISA) was developed using specific polyclonal anti-apalbumin 1 antibody. The limit of detection for apalbumin 1 was 2 ng mL(-1). The floral honeys contained apalbumin 1 as follows: acacia, 0.011%; linden, 0.010%; chestnut, 0.029%; rape, 0.010%; and dandelion, 0.014%. The saccharose syrup honey contained only 0.001% of apalbumin 1. The average amount of apalbumin 1 relating to the total protein content of analyzed honey samples was 23.39%, whereas in SCCH apalbumin 1 presented only 4.81% of total proteins of honey. Apalbumin 1 is thermostabile in honey at 80 degrees C incubated for 40 min. ELISA results show good precision in the evaluation of apalbumin 1 quantity in honey (CV ranged from 0.69 to 4.25%).

Towards functional proteomics of minority component of honeybee royal jelly: the effect of post-translational modifications on the antimicrobial activity of apalbumin2.

This study illustrates multifunctionality of proteins of honeybee royal jelly (RJ) and how their neofunctionalization result from various PTMs of maternal proteins. Major proteins of RJ, designated as apalbumins belong to a protein family consisting of nine members with M(r) of 49-87 kDa and they are accompanied by high number of minority homologs derived from maternal apalbumins. In spite of many data on diversity of apalbumins, the molecular study of their individual minority homologous is still missing. This work is a contribution to functional proteomics of second most abundant protein of RJ apalbumin2 (M(r) 52.7 kDa). We have purified a minority protein from RJ; named as apalbumin2a, differ from apalbumin2 in M(r) (48.6 kDa), in N-terminal amino acids sequences - ENSPRN and in N-linked glycans. Characterization of apalbumin2a by LC-MALDI TOF/TOF MS revealed that it is a minority homolog of the major basic royal jelly protein, apalbumin2, carrying two fully occupied N-glycosylation sites, one with high-mannose structure, HexNAc2Hex9, and another carrying complex type antennary structures, HexNAc4Hex3 and HexNAc5Hex4. We have found that apalbumin2a inhibit growth of Paenibacillus larvae. The obtained data call attention to functional plasticity of RJ proteins with potential impact on functional proteomics in medicine.

Isolation and characterization of chitin from bumblebee (Bombus terrestris).

Insect chitin possessing shell-like structure was prepared from the bumblebee corpses by a consequent treatment with 1M HCl and 1M NaOH. The bumblebee chitin was compared with crustacean (shrimp) chitin by using elemental analysis, Fourier-transform infrared (FT-IR) and solid-state (13)C cross-polarization magic angle spinning nuclear magnetic resonance (CP/MAS)-NMR spectroscopy and confocal microscopy. Both chitins (bumblebee and shrimp) exhibited identical spectra, while the bumblebee chitin had a 5% lower degree of acetylation and was characterized by a fine membrane texture.

The immunostimulatory effect of the recombinant apalbumin 1-major honeybee royal jelly protein-on TNFalpha release.

Apalbumin1 (Apa1) is the major royal jelly (RJ) and honey glycoprotein having various biological properties. We have previously demonstrated that Apa1 is a regular component of honey and honeybee pollen and stimulates macrophages to release tumor necrosis factor alpha (TNFalpha). The recombinant Apa1 (rApa1) and its four recombinant protein fragments derived on the basis of partial tryptic products of Apa1 were prepared by heterologous expression in Escherichia coli BL21-CodonPlus(DE3)-RIL. L-arginine at 50 mM concentration was used for improving the recombinant protein solubility. We report that the proteinous moiety of glycoprotein is responsible for stimulation of TNFalpha production by murine peritoneal macrophages. Moreover, we have shown that immunostimulatory effect is significantly increased after partial tryptic digestion of Apa1. It has been determined that recombinant N-terminal fragment of Apa1 is the most active elicitor of TNFalpha release in comparison to other three protein fragments of Apa1, as well as to the native Apa1 and rApa1. Furthermore, it was found that native honey was able to stimulate TNFalpha secretion from murine macrophages, whereas the deproteinized honey had no effect on the release of TNFalpha. This result suggests that immunostimulatory effect of honey is based on its RJ-protein content, primarily on its dominant protein Apa1.

Stimulation of TNF-alpha release by fungal cell wall polysaccharides.

Carboxymethylated derivatives were prepared from the (1-->3)-beta-D-glucan isolated from the cell wall of baker's yeast Saccharomyces cerevisiae and from the chitin-glucan complex of the mycelium of the industrial filamentous fungus Aspergillus niger. The polysaccharides were applied to peritoneal mouse macrophages and after a 2-h incubation the release of TNF-alpha by the stimulated macrophages was measured using an enzyme-linked immunosorbent assay. As the third polysaccharide stimulant, a water-soluble derivative of chitin was assayed and the observed cytokine release was compared with the control experiment. In three concentrations of the polysaccharides applied, carboxymethyl glucan revealed a dramatic increase in the TNF-alpha release, while addition of carboxymethyl chitin-glucan resulted only in a moderate enhancement, and carboxymethyl chitin was inactive. The results indicate that fungal polysaccharides, especially (1-->3)-beta-D-glucan, are potent macrophage stimulators and activators of TNF-alpha release, which implies their potential application in antitumor therapy.

Two structurally different defensin genes, one of them encoding a novel defensin isoform, are expressed in honeybee Apis mellifera.

Two defensins showing high mutual similarity have previously been characterized in honeybee Apis mellifera: royalisin, a peptide isolated from the royal jelly, and defensin, found in the hemolymph of bacterially infected bees. Here we show that both these peptides are encoded by the same polymorphic gene, which we termed defensin1. Besides this gene, we identified an additional defensin gene coding for a novel honeybee defensin designated defensin2. The pre-pro-peptide sequence of defensin 2 was inferred from its cDNA. Mature defensin 2 peptide shows 55.8% identity with defensin 1. Sequences of genomic loci of the two defensin genes revealed their different structure. Defensin1 possesses an exon-intron structure unique among arthropoda defensin genes. Its second intron splits exactly the common structural module of defensins from a short amidated C-terminal extension found only in hymenopteran defensins. Transcription of defensin genes in some nurse honeybees tissues was studied by RT-PCR. Both defensins are expressed in heads and thoraces. Defensin1 but not defensin2 mRNA was detected in hyphopharyngeal, mandibular and thoracic salivary glands. Immune response elements were identified by computer analysis of the promoter regions of defensin genes. Their different representation in these genes reflects presumably observed tissue-specific expression of defensins.

Immunochemical approach to detection of adulteration in honey: physiologically active royal jelly protein stimulating TNF-alpha release is a regular component of honey.

The presence of royal jelly (RJ) proteins in honey collected from nectars of different plants, origin, and regions and in honeybee's pollen was detected by Western-blot analysis using polyclonal antibodies raised against water-soluble RJ-proteins. The most abundant RJ-protein in honeybee products corresponded to a 55 kDa protein. The N-terminal amino acid sequence of 55 kDa protein was N-I-L-R-G-E. This sequence is identical to the apalbumin-1, the most abundant protein of RJ. Apalbumin-1 is a regular component of honeybee products and thus is a suitable marker tool for proving adulteration of honey by means of immunochemical detection. Its presence in all tested samples of honeys and honeybee pollen was confirmed also by Western-blot analysis using polyclonal antibodies raised against recombinant apalbumin-1. It has been found that major RJ-proteins, apalbumin-1, and apalbumin-2, stimulate mouse macrophages to release TNF-alpha, which demonstrates that physiologically active proteins of honey could be used for its biological valuation.

Honeybee (Apis mellifera L.) mrjp gene family: computational analysis of putative promoters and genomic structure of mrjp1, the gene coding for the most abundant protein of larval food.

Mrjp1 gene belongs to the honeybee mrjp gene family encoding the major royal jelly proteins (MRJPs), secreted by nurse bees into the royal jelly. In this study, we have isolated the genomic clone containing the entire mrjp1 gene and determined its sequence. The mrjp1 gene sequence spans over 3038 bp and contains six exons separated by five introns. Seven mismatches between the mrjp1 gene sequence and two previously independently published cDNA sequences were found, but these differences do not lead to any change in the deduced amino acid sequence of MRJP1. With the aid of inverse polymerase chain reaction we obtained sequences flanking the 5' ends of other mrjp genes (mrjp2, mrjp3, mrjp4 and mrjp5). Putative promoters were predicted upstream of all mrjp genes (including mrjp1). The predicted promoters contain the TATA motif (TATATATT), highly conserved both in sequence and position. Ultraspiracle (USP) transcription factor (TF) binding sites in putative promoter regions and clusters of dead ringer TF binding sites upstream of these promoters were predicted computationally. We propose that USP, as a juvenile hormone (JH) binding TF, might possibly act as a mediator of mrjp expression in response to JH. Mrjp1's genomic locus is predicted to encode an antisense transcript, partially overlapping with five mrjp1 exons and entirely overlapping with the putative promoter and predicted transcriptional start point of mrjp1. This finding may shed light on the mechanisms of regulation of mrjps expression. Southern blot analysis of genomic DNA revealed that all so far known members of mrjp gene family (mrjp1, mrjp2, mrjp3, mrjp4 and mrjp5) are present as single-copy genes per haploid honeybee genome. Although MRJPs and the yellow protein of Drosophila melanogaster share a certain degree of similarity in aa sequence and although it has been shown that they share a common evolutionary origin, neither structural similarities in the gene organization, nor significant similarities between intron sequences of mrjp1 gene and fourteen yellow-like genes of D. melanogaster were found.