Unique Molecular Architecture of Silk Fibroin in the Waxmoth,Galleria mellonella

Abstract

Proteins of silk fibers are characterized by reiterations of amino acid repeats. Physical properties of the fiber are determined by the amino acid composition, the complexity of repetitive units, and arrangement of these units into higher order arrays. Except for very short motifs of 6 -10 residues, the length of repetitive units and the number of these units concatenated in higher order assemblies vary in all spider and lepidopteran silks analyzed so far. This paper describes an exceptional silk protein represented by the 500-kDa heavy chain fibroin (H-fibroin) of the waxmoth, Galleria mellonella. Its non-repetitive N-terminal (175 residues) and C-terminal (60 residues) parts, the overall gene organization, and the nucleotide sequence around the TATA box show that it is homologous to the H-fibroins of other Lepidoptera. However, over 95% of the protein consists of highly ordered repetitive structures that are unmatched in other species. The repetitive region includes 11 assemblies AB 1 AB 1 AB 1 AB 2 (AB 2 )AB 2 of remarkably conserved polypeptide repeats A (63 amino acid residues), B 1 (43 residues), and B 2 (18 residues). The repeats contain a high proportion of Gly (31.6%), Ala (23.8%), Ser (18.1%), and of residues with long hydrophobic side chains (16% for Leu, Ile, and Val combined). The presence of the GLGGLG and SSAASAA(AA) motifs suggests formation of pleated ␤-sheets and their stacking into crystallites. Conspicuous conservation of the apolar sequence VIVI followed by DD or ED is interpreted as indicating the importance of hydrophobicity and electrostatic charge in H-fibroin cross-linking. The environment of G. mellonella larvae within bee cultures requires continuous production of silk that must be both strong and elastic. The spectacular arrangement of the repetitive H-fibroin region apparently evolved to meet these requirements.

Correlation between Fibroin Amino Acid Sequence and Physical Silk Properties

“…The introns are alike by having high contents of adenine and thymidine, but homology is restricted to the exon/intron and intron/exon boundaries demarcated by the classical GT/AG borders. The intron in G. mellonella H-fibroin is unusually long due to insertion of a repetitive DNA element, Gm1 (9). The beginning of the second exon exhibits a high degree of interspecies homology; the first 326 nt in G. mellonella, 323 nt in E. kuehniella, and 320 nt in P. intepunctella can be aligned with just a few gaps that represent occasional deletions or insertions (Fig.…”

“…The dominant RT-PCR products obtained with the E1/R1 pair were about 1000 nt long in E. kuehniella and 750 nt in P. interpunctella. The similarity of their sequences with the G. mellonella H-fibroin gene (9) left no doubt that they represented 5Ј ends of H-fibroin cDNAs of the respective species. The PCR with genomic DNA template and primers E1 and R2 resulted in amplification of multiple products ranging in size from about 500 to 2500 nt.…”

“…The remarkable homogeneity of the repeat length and composition contrasted with repeat variability in the H-fibroins of Bombyx and Antheraea (6 -8). Repeat regularity, absence of concatenations of short motifs, and relatively high content of amino acid residues with bulky side chains indicated that organization of the H-fibroin polymer in G. mellonella differs from that in Bombyx and Antheraea (9).…”

“…The silk produced by G. mellonella must obviously be extensible to allow widening of the tube when the larva turns around and at the same time strong enough to hold the larval weight. It was deduced that the uniformity of H-fibroin repeats underlies the excellent mechanical properties of G. mellonella silk (9).…”

“…On the basis of x-ray diffraction data (1), we expected to find polyalanine repeats in the H-fibroin of the waxmoth Galleria mellonella (family Pyralidae), but gene analysis revealed a more complex amino acid arrangement (9). The overall gene structure and the nucleotide sequences at the 5Ј and 3Ј gene ends were similar to the H-fibroin genes of B. mori (6), A. pernyi (7), and Antheraea yamamai (8), but the major internal sequence was unique.…”