Jozef Šimúth

A FAMILY OF MAJOR ROYAL JELLY PROTEINS OF THE HONEYBEE APIS MELLIFERA L.

Abstract. The characterization of major proteins of honeybee larval jelly (49 – 87 kDa) was performed by the sequencing of new complementary DNAs (cDNAs) obtained from a honeybee head cDNA library, by the determination of N-terminal sequences of the proteins, and by analyses of the newly obtained and known sequence data concerning the proteins. It was found that royal jelly (RJ) and worker jelly (WJ) contain identical major proteins and that all the proteins belong to one protein family designated MRJP (from major royal jelly proteins). The family consists of five main members (MRJP1, MRJP2, MRJP3, MRJP4, MRJP5). The proteins MRJP3 and MRJP5 are polymorphic. MRJPs account for 82 to 90% of total larval jelly protein, and they contain a relatively high amount of essential amino acids. These findings support the idea that MRJPs play an important role in honeybee nutrition.

The Family of Major Royal Jelly Proteins and Its Evolution

Abstract. A cDNA encoding a new member of the gene family of major royal jelly proteins (MRJPs) from the honeybee, Apis mellifera, was isolated and sequenced. Royal jelly (RJ) is a secretion of the cephalic glands of nurse bees. The origin and biological function of the protein component (12.5%, w/w) of RJ is unknown. We show that the MRJP gene family encodes a group of closely related proteins that share a common evolutionary origin with the yellow protein of Drosophila melanogaster. Yellow protein functions in cuticle pigmentation in D. melanogaster. The MRJPs appear to have evolved a novel nutritional function in the honeybee.

Apisimin, a new serine–valine-rich peptide from honeybee (Apis mellifera L.) royal jelly: purification and molecular characterization1

A peptide named apisimin was found in honeybee (Apis mellifera L.) royal jelly (RJ). N‐terminal sequencing showed that this peptide corresponded to the sequence of a cDNA clone isolated from an expression cDNA library prepared from heads of nurse honeybees. No homology was found between the protein sequence of apisimin with a molecular mass of 5540.4 Da and sequences deposited in the Swiss‐Prot database. The 54 amino acids of apisimin do not include Cys, Met, Pro, Arg, His, Tyr, and Trp residues. The peptide shows a well‐defined secondary structure as observed by CD spectroscopy, and has the tendency to form oligomers. Isoelectrofocusing showed apisimin to be an acidic peptide.

Molecular characterization of MRJP3, highly polymorphic protein of honeybee (Apis mellifera) royal jelly

Major proteins of honey bee (Apis mellifera) royal jelly are members of the MRJP protein family. One MRJP protein termed MRJP3 exhibits a size polymorphism as detected by SDS-PAGE. In this report we show that polymorphism of the MRJP3 protein is a consequence of the polymorphism of a region with a variable number of tandem repeats (VNTR) located at the C-terminal part of the MRJP3 coding region. We present the characterization of five polymorphic alleles of MRJP3 by DNA sequencing. By PCR analyses, at least 10 alleles of distinct sizes were found in randomly sampled bees. Studies with nurse bees from a single honeybee colony revealed both Mendelian inheritance and very high variability of the MRJP3 genomic locus. The high variability and simple detection of the MRJP3 polymorphism may be useful for genotyping of individuals in studies of the honeybee.

Molecular cloning of two cDNAs from the head of the nurse honey bee (Apis mellifera L.) for coding related proteins of royal jelly

SUMMARYTwo cDNA clones designated pRJP57–1 and pRJP57–2 which code for related polypeptides of major royal jelly protein were identified from the λUni-ZAP XR library of nurse honey bee head cDNA. The nucleotide sequences of the cDNA inserts show a 70.2% identity. From the open reading frames of pRJP57–1 and pRJP57–2 the amino acid sequences for polypeptides of 467 and 464 amino acid residues were derived, showing an identity of 56.6%. No significant homology with hitherto known proteins was observed. Both proteins contain N-glycosylation sites, phosphorylation sites for three types of protein kinases and almost identical signal peptides. Northern analysis showed that total mRNA contains a high proportion of RJP57–1 and RJP57–2 mRNAs (8% and 2% respectively).

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%).

Stimulation of TNF-α Release by Fungal Cell Wall Polysaccharides

Carboxymethylated derivatives were prepared from the (1→3)-β-ᴅ-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-α 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-α 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)-β-ᴅ-glucan, are potent macrophage stimulators and activators of TNF-α release, which implies their potential application in antitumor therapy.

Production and purification of Japanese quail ovalbumin as fusion protein with glutathione S-transferase in Escherichia coli.

A plasmid encoding a fusion protein interlinked by thrombin recognition sequence between glutathione S-transferase and Japanese quail ovalbumin (without 40 amino acid residues from the 5′-end of the ORF) has been constructed, employing the expression system pGEX-2T. The deglycosylated fusion protein (64 kDa) was purified by affinity chromatography on glutathione agarose beads, analyzed by SDS-polyacrylamide gel electrophoresis, immunochemically detected with antiserum raised against Japanese quail ovalbumin and tested for its stability.