Carbonic anhydrase generates a pH gradient in Bombyx mori silk glands

Abstract

Silk is a protein of interest to both biological and industrial sciences. The silkworm, Bombyx mori, forms this protein into strong threads starting from soluble silk proteins using a number of biochemical and physical cues to allow the transition from liquid to fibrous silk. A pH gradient has been measured along the gland, but the methodology employed was not able to precisely determine the pH at specific regions of interest in the silk gland. Furthermore, the physiological mechanisms responsible for the generation of this pH gradient are unknown. In this study, concentric ion selective microelectrodes were used to determine the luminal pH of Bombyx mori silk glands. A gradient from pH 8.2 to 7.2 was measured in the posterior silk gland, with a pH 7 throughout the middle silk gland, and a gradient from pH 6.8 to 6.2 in the beginning of the anterior silk gland where silk processing into fibers occurs. The small diameter of the most anterior region of the anterior silk gland prevented microelectrode access in this region. Using a histochemical method, the presence of active carbonic anhydrase was identified in the funnel and anterior silk gland of fifth instar larvae. The observed pH gradient collapsed upon addition of the carbonic anhydrase inhibitor methazolamide, confirming an essential role for this enzyme in pH regulation in the Bombyx mori silk gland. Plastic embedding of whole silk glands allowed clear visualization of the morphology, including the identification of four distinct epithelial cell types in the gland and allowed correlations between silk gland morphology and silk stages of assembly related to the pH gradient. Bombyx mori silk glands have four different epithelial cell types, one of which produces carbonic anhydrase. Carbonic anhydrase is necessary for the mechanism that generates an intraluminal pH gradient, which likely regulates the assembly of silk proteins and then the formation of fibers from soluble silk proteins. These new insights into native silk formation may lead to a more efficient production of artificial or regenerated silkworm silk fibers.

Changes in Silk Feedstock Rheology during Cocoon Construction: The Role of Calcium and Potassium Ions

“…Nevertheless, the present work revealed no direct correlation between the hemolymph pH and the measured viscosity, as shown in Figure b. Moreover, the pH of the feedstock appeared to remain roughly neutral (6.5 to 7.2), in agreement with previous work and suggesting that the gland contents are insulated from changes in the hemolymph.…”

“…Based on the published amino acid sequence, fibroin is believed to contain small proportions of phenolic, carboxylic acid, or amine substituents in the side‐groups (≈5.2, 1.4, and 0.9 mol%, respectively). Previous work has suggested that interactions between these groups are sensitive to pH, thereby affecting the fibroin chain conformation and viscosity . Indeed, rheological changes brought about by exposing silk feedstock to ethanoic acid or ammonia vapors have previously been reported by Terry et al …”

Interfacial Behavior of Recombinant Spider Silk Protein Parts Reveals Cues on the Silk Assembly Mechanism

“…[8][9][10][11] A factor known to regulate silk assembly is the decrease in pH along the duct, caused by carbanhydrase production and proton pumps. [12][13][14] It is also suggested that ion concentration gradients, dehydration of the protein dope, and hydrodynamic forces that increase along the narrowing duct facilitate the fiber formation. 4,11,[15][16][17] .…”

Aquatic caddisworm silk is solidified by environmental metal ions during the natural fiber‐spinning process

“…As stored in the gland lumen, H-fibroins lack b-sheet secondary structure, occupying instead mostly type II b-turn and random coil conformations, a form referred to as silk I (12,13). At the relatively high pH (6.9-8.0) in the posterior silk gland lumen, like-charge repulsion between the negatively charged termini and short, negatively charged spacers interspersed in the poly(GA) central region is thought to maintain the silk I conformation and the colloidal fluid form of the silk complexes (11,14,15). Fiber formation begins as the fluid silk dope is sheared through the tapered anterior gland with a coincident drop in pH from 6.9 to 4.8 (11).…”