Molecular architecture of silk fibroin of Indian golden silkmoth, Antheraea assama

Silk is a remarkable proteinaceous biomaterial, which is a unique possession of arthropods. Though silks are produced for an enormous number of purposes, holometabolous insects secrete a silken cocoon to encase their metamorphosing pupae, as a significantly strong selection factor. The cocoon silk of the domesticated silkworm, Bombyx mori, is globally renowned and its principal protein, fibroin is extensively studied. X-ray diffraction studies showed the presence of β-sheets in fibroin that are formed by the stacking of reiterated short arrays composed of small amino acids1,2. Lepidopteran larvae secrete silk from a pair of tubular secretory glands called silk glands, which are demarcated into posterior (PSG), middle (MSG) and anterior (ASG) regions that exit through a spinning orifice3. B. mori silk fiber has a fibrous core made of three elementary polypeptides, a fibroin heavy chain (H-fibroin or Fhc) of ~390 kDa, a fibroin light chain (L-fibroin or Flc) of 30 kDa which makes heterodimers and six such dimers interact with a glycoprotein, P25 to form 2.3 MDa elementary structural units of the fibrous core of silk, which in turn is multiply tunicated with glue proteins called sericins4,5,6,7. However, the fibrous core of silk secreted by wild silkmoths (family Saturniidae) is solely composed of H-fibroin with stretches of polyalanine that makes homodimers2,8. Based on X-ray diffraction studies, silk fibers characterized by the presence of strings of alanine were classified under group 3a with an intersheet packing of 10.6 A9. Indian golden silkmoth, Antheraea assama (family Saturniidae) is semi-domesticated with a narrow habitat range confined to Brahmaputra valley of northeast India. A. assama is commonly called muga silkworm which spins golden cocoon silk, culturally acclaimed as a special product of India and the most expensive of silks10. It is highly valued in textile industry and in designing novel biomaterials for its unique biophysical properties like golden luster, tenacity and high absorbance of UV radiation11,12.However, extensive rearing and prospects of global recognition are deterred by the moth’s semi-domestic nature and extremely confined geographical distribution. As the major component of silk fiber, the structure of H-fibroin determines its physical properties, which in turn are dictated by the type of the composite amino acids and their pattern of arrangement in full length. Determining full length gene sequence is significant to understand the role of each protein structural unit in the big picture. The sequences responsible for specific properties of interest allow engineering of better chimeric genes to refine the biophysical properties of fiber to spin composite silk fibers with better mechanical properties and to overcome the problems of endogenously expressed wild silks13. Sequence data of complex genes like H-fibroin allows the understanding of its relative status among similar genes and its adaptive trajectory in evolution. They also are important models of study for unusual evolutionary events like genetic polymorphism and accumulation of repetitive units by duplication through unequal crossing-over14. The similarity in evolution of repetitive region with that of the microsatellites evolution could be responsible for their clonal instability, making it formidable to characterize the complete structure of a full length H-fibroin8. In order to explain the genetic and biochemical factors responsible for its properties, this report describes the comprehensive structure and expression of A. assama fibroin (AaFhc) gene that encodes H-fibroin protein. Silk gland specific transcriptomic sequence data from WildSilkBase EST library and in-house generated cDNA library were used to identify partial sequences of H-fibroin15. Full length H-fibroin gene was amplified through genomic PCR based on terminal sequence conservation and was cloned for determination of the complete sequence by constructing a sub-clone library of unique loci derived from separate digestions with different restriction endonucleases. The full length sequence was analysed in silico, to determine bias in usage of isocodons of its major amino acid residues, their composition in conceptually translated coding sequence, motif-assembly and fine repetitious organization of these motifs to predict secondary structure responsible for its remarkable properties and to study the evolutionary divergence of AaFhc from other H-fibroins. In addition, the report also describes the structure of A. assama silk gland and its cell enumeration details.