Spider egg case core fibers: trimeric complexes assembled from TuSp1, ECP-1, and ECP-2.
Spider silk proteins are well-known for their extraordinary mechanical properties, displaying remarkable strength and toughness. In this study, matrix-assisted laser desorption ionization (MALDI) tandem time-of-flight (TOF) mass spectrometry (MS/MS) and reverse genetics were used to isolate a new cDNA sequence that encodes for a protein assembled into egg case silk from the black widow spider, Latrodectus hesperus. Analysis of the primary sequence of this protein reveals approximately 52% identity to the egg case protein 1 (ECP-1) fibroin-like family member. On the basis of the similarity in the primary sequence and expression pattern, we have named this factor egg case protein 2 (ECP-2). Alignments of ECP-1 and ECP-2 demonstrate highly conserved N termini, with 16 Cys residues found within the first 153 amino acids. Traditional ensemble repeats found within reported fibroins were poorly represented in the primary sequence of ECP-2, but scattered blocks of polyalanine were present, along with a C terminus rich in GA repeats. Reverse transcription quantitative PCR analysis showed that ECP-2 is predominantly expressed in the tubuliform gland. Relative to ECP-1, ECP-2 mRNA levels were determined to be >2-fold higher. MALDI MS/MS analysis of peptide fragments generated from the large-diameter core fiber after enzymatic digestion and acid hydrolysis demonstrated the presence of a fiber that is trimeric in nature, containing tubuliform spidroin 1 (TuSp1), ECP-1, and ECP-2. We also report an additional primary sequence for TuSp1, demonstrating that TuSp1 contains two Cys residues within a nonrepetitive N-terminal region. In combination with the distinctive protein architectures of ECP-1 and ECP-2, along with their co-localization with TuSp1 in the core fiber, our findings suggest that ECP-1 and ECP-2 play important structural roles in the egg case silk fiber.
Department of Chemistry, University of the Pacific, Stockton, California 95211, USA.
Xiaoyi
X
Hu
Kristin
K
Kohler
Arnold M
AM
Falick
Anne M F
AM
Moore
Patrick R
PR
Jones
Craig
C
Vierra
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001740:Black Widow Spider / Q000737:chemistry / Q000196:embryology / Q000378:metabolism*; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry; D005349:Fibroins / Q000737:chemistry / Q000378:metabolism; D015723:Gene Library; D019476:Insect Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010063:Ovum / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018076:DNA, Complementary; D019476:Insect Proteins; D020033:Protein Isoforms; D005349:Fibroins; D003545:Cysteine