title: 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 B. 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 B. 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. B. 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.
authors.affiliation: Department of Biomedical Engineering, Tufts University, Medford, MA, USA; School of Biological Sciences, The University of Auckland, Auckland, New Zealand.
authors.forename: L J
authors.initials: LJ
authors.lastname: Domigan
authors.affiliation: Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Andersson
authors.affiliation: Department of Biomedical Engineering, Tufts University, Medford, MA, USA.
authors.forename: K A
authors.initials: KA
authors.lastname: Alberti
authors.affiliation: Department of Neurosurgery, Physiology and Neuroscience, New York University School of Medicine, New York, NY, USA.
authors.forename: M
authors.initials: M
authors.lastname: Chesler
authors.affiliation: Department of Biomedical Engineering, Tufts University, Medford, MA, USA.
authors.forename: Q
authors.initials: Q
authors.lastname: Xu
authors.affiliation: Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden; Department of Neurobiology, Care Sciences and Society, Karolinska Institute, Huddinge, Sweden; Institute of Mathematics and Natural Sciences, Tallinn University, Tallinn, Estonia.
authors.forename: J
authors.initials: J
authors.lastname: Johansson
authors.affiliation: Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden; Department of Neurobiology, Care Sciences and Society, Karolinska Institute, Huddinge, Sweden. Electronic address: anna.rising@slu.se.
authors.forename: A
authors.initials: A
authors.lastname: Rising
authors.affiliation: Department of Biomedical Engineering, Tufts University, Medford, MA, USA. Electronic address: david.kaplan@tufts.edu.
authors.forename: D L
authors.initials: DL
authors.lastname: Kaplan
mesh_terms: D000818:Animals; D012831:Bombyx / Q000201:enzymology*; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000378:metabolism*; D005088:Exocrine Glands / Q000166:cytology / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007814:Larva / Q000201:enzymology; D008704:Methazolamide / Q000494:pharmacology; D018892:Proton-Motive Force; D047011:Silk / Q000096:biosynthesis
chemical_list: D002257:Carbonic Anhydrase Inhibitors; D047011:Silk; D002256:Carbonic Anhydrases; D008704:Methazolamide
keywords: Carbonic anhydrase; Fibroin; Ion-selective microelectrodes; Morphology; Silk gland