Pyriform spidroin 1, a novel member of the silk gene family that anchors dragline silk fibers in attachment discs of the black widow spider, Latrodectus hesperus

Abstract

Spiders spin high performance threads that have diverse mechanical properties for specific biological applications. To better understand the molecular mechanism by which spiders anchor their threads to a solid support, we solubilized the attachment discs from black widow spiders and performed in-solution tryptic digests followed by MS/MS analysis to identify novel peptides derived from glue silks. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and cDNA library screening, we isolated a novel member of the silk gene family called pysp1 and demonstrate that its protein product is assembled into the attachment disc silks. Alignment of the PySp1 amino acid sequence to other fibroins revealed conservation in the non-repetitive C-terminal region of the silk family. MS/MS analysis also confirmed the presence of MaSp1 and MaSp2, two important components of dragline silks, anchored within the attachment disc materials. Characterization of the ultrastructure of attachment discs using scanning electron microscopy studies support the localization of PySp1 to small diameter fibers embedded in a glue-like cement, which network with large diameter dragline silk threads, producing a strong, adhesive material. Consistent with elevated PySp1 mRNA levels detected in the pyriform gland, MS analysis of the luminal contents extracted from the pyriform gland after tryptic digestion support the assertion that PySp1 represents one of the major constituents manufactured in the pyriform gland. Taken together, our data demonstrate that PySp1 is spun into attachment disc silks to help affix dragline fibers to substrates, a critical function during spider web construction for prey capture and locomotion.

FIGURE 1.

Physical structure of an attachment disc collected from a black widow spider. A, black widow spiders use attachment disc material (black arrow) to anchor dragline silk to substrates to facilitate locomotion and web-building functions. This image was captured with a Canon EOS 40D digital camera. B, SEM of an attachment disc at ×250 magnification; white arrow denotes dragline silk fibers leading into attachment disc. C, SEM of an attachment disc at ×900. D, SEM of an attachment disc at ×1500; red arrow represents an attachment disc fiber.

FIGURE 2.

MALDI-TOF tandem analyses of precursor peptide ions generated from an in-solution tryptic digestion of solubilized attachment disc materials. A, high energy collision-activated dissociation spectrum of precursor ion with m/z 986.5 (MH⁺, monoisotopic). The sequence of this peptide was found to be AQAQAEAAR. B, high energy collision-activated dissociation spectrum of precursor ion with m/z 1563.8. This peptide ion had the derived sequence LASLSQAMSSVLSGGR. The N- or C-terminal leucine residues could be any combination of leucine and/or isoleucine. The middle leucine residue was confirmed with the existence of the w13 ion.

FIGURE 3.

The amino acid sequence of L. hesperus PySp1 shows similarity to spider fibroin family members. A, translation of the nucleotide sequence from the PySp1 cDNA contains an ORF. Peptide sequences determined by MS/MS analysis (in-solution tryptic digest products from LiBr solubilized attachment disc silks) that are found within the ORF are underlined. Red and blue denote the block repeats with either the consensus AAARAQAQAEARAKAE or AAARAQAQAE. The last 122 amino acids represent the non-repetitive C terminus. B, alignment of the PySp1 C-terminal sequence reveals similarity to other fibroin family members. Alignments were performed with the last C-terminal 122 amino acids of the fibroins using the computer algorithm CLUSTALW. Amino acids are represented by one-letter abbreviations, gaps are indicated by dashes. Shaded gray areas represent residues that are found in the highest frequency at that position for the fibroin family members. The blue underlined region represents a segment that is 100% identical to the peptide ion at m/z 1563.8; this region is unique to PySp1. Purple denotes a 78-amino acid region that is rich in histidine and other polar residues. GenBank accession codes for the L. hesperus fibroin family members are listed: MaSp1 (DQ409057), MaSp2 (DQ409058), MiSp1-like (EU394445.1), PySp1 (FJ973621), AcSp1-like (EU025854.1), and TuSp1 (DQ109035). C, maximum likelihood tree of the nucleotide sequence encoding the conserved, non-repetitive C-terminal region of spider fibroin genes from all known members of the gene family except MiSp2 using the codon model of Goldman and Yang (29) with F3 × 4 codon frequencies. The model of evolution used was GTR+G. PySp1 apparently arose through duplication of the ancestral gene that also gave rise to tusp genes. Although this placement is not robust to bootstrapping, the same phylogenetic arrangement was found when the data were analyzed by an amino acid model (WAG+I+G+F). D, predicted amino acid composition profile from the translated cDNAs L. hesperus MaSp1 (EF595246.1), MaSp2 (EF595245.1), TuSp1 (AY953070.1), MiSp1-like (EU394445.1), AcSp1-like (EU025854.1), and PySp1 (FJ973621). The translated gene sequences used for MaSp1 and MaSp2 represent full-length protein sequences, whereas TuSp1, MiSp1-like, AcSp1-like, and PySp1 represent partial protein sequences.

FIGURE 4.

Luminal contents of the pyriform gland contain proteins with large amounts of alanine and glutamine. Proteins stored in the lumen of the seven different silk-producing glands from L. hesperus were extracted and subjected to acid hydrolysis. A, major ampullate; B, minor ampullate; C, flagelliform; D, aggregate; E, aciniform; F, tubuliform; and G, pyriform. Data from the major and minor ampullate glands, as well as the aciniform gland were previously determined (6, 12).

FIGURE 5.

Pyriform glands are fingerlike structures that express PySp1 mRNA at high levels. A, real time quantitative PCR analysis was used to determine the mRNA expression pattern of the novel fibroin in a variety of different tissues. Total RNA was isolated from the major ampullate gland (MA), minor ampullate gland (MI), tubuliform (TB), flagelliform (FL), aciniform (AC), pyriform (PY), aggregate (AG), and fat tissues (FT). Equivalent amounts of total RNA were reversed transcribed using Superscript III and aliquots used for quantitative reverse transcriptase-PCR. Reactions were performed in triplicate and normalized internally using the black widow actin mRNA. Data are representative of experimental results obtained from two independent trials. B, pyriform glands were dissected from black widow spiders (upper right, pyriform gland removed from a single spider and photographed on a Leica MZ16 dissecting microscope at ×10 magnification; lower left, aciniform tissue is also shown in the field of view; fat tissue is surrounding both glands).

FIGURE 6.

MALDI-TOF mass spectrum of an in-solution trypsin digestion of luminal contents extracted from the pyriform gland of L. hesperus. Peptide ions from the protein PySp1 are labeled with #, with the exception of peptide ion 995.5. Trypsin autolysis peptide signals are labeled with an asterisk. The spectrum was recorded using a CHCA matrix and the instrument was operated in reflectron mode.