Abstract
The primary structure of the alpha-chain of preCol-D (molecular mass = 80 kDa), a tanned collagenous protein predominating in the distal portion of the byssal threads of the mussel Mytilus edulis, was deduced from cDNA to encode an unprecedented natural block copolymer with three major domain types: a central collagen domain flanked by fibroin-like domains and followed by histidine-rich termini. The fibroin-like domains have sequence motifs that strongly resemble the crystalline polyalanine-rich and amorphous glycine-rich regions of spider dragline silk fibroins. The terminal regions resemble the histidine-rich domains of a variety of metal-binding proteins. The silk domains may toughen the collagen by increasing its strength and extensibility. PreCol-D expression is limited to the mussel foot, which contains a longitudinal gradient of preCol-D mRNA. This gradient increases linearly in the proximal to distal direction and reaches a maximum just before the distal depression of the foot.
Highlights
EXPERIMENTAL PROCEDURESPartial Characterization of PreCol-D—Common mussels M. edulis were collected at Union, Maine, or Lewes, Delaware
The primary structure of the ␣-chain of preCol-D, a tanned collagenous protein predominating in the distal portion of the byssal threads of the mussel Mytilus edulis, was deduced from cDNA to encode an unprecedented natural block copolymer with three major domain types: a central collagen domain flanked by fibroin-like domains and followed by histidine-rich termini
A number of amino acid sequences were determined from intact and protease-digested preCol-D to establish a basis for comparing cDNA sequences with the protein isolated from the foot as well as from byssal threads
Summary
Partial Characterization of PreCol-D—Common mussels M. edulis were collected at Union, Maine, or Lewes, Delaware. The second extraction contains a stable partially degraded intermediate of preCol-D (apparent molecular mass 70 kDa), which was purified by chromatography on Sephadex G-200 (eluted with 5% acetic acid), followed by C-4 reversed phase (column 250 x 7-mm; Applied Biosystems, Foster City, CA) HPLC eluted with a gradient (28 – 48% acetonitrile and 0.1% trifluorocetic acid) [1]. Fragments were purified by C-4 HPLC using a linear acetonitrile gradient (42–56% over 60 min) with 0.1% v/v trifluoroacetic acid and partially sequenced. Several peptides were produced by clostridial collagenase digestion of Col-D at a protein-to-enzyme ratio of 100:1. These were purified by C-18 reversed phase HPLC using a linear gradient of acetonitrile (0 –22% over 50 min) with 0.1% trifluoroacetic acid and sequenced [1, 8]. One additional extraction of Col-D was carried out on byssal threads
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