Purification and physical properties of nematode mini-titins and their relation to twitchin

RUDIGER NAVE 0 1 DIETER FURST 0 1 UWE VINKEMEIER 0 1 KLAUS WEBER 0 1 0 Present address: Byk Gulden , Postfach 6500, D-7750 Konstanz, FRG 1 Max Planck Institute for Biophysical Chemistry, Department of Biochemistry , P.O. Box 2841, D-3400 Goettingen, FRG relation to twitchin - We have isolated mini-titin from the nematodes Ascaris lumbricoides and Caenorhabditis elegans under native conditions using a modification in the procedure to prepare this protein from insect muscle. The proteins have an apparent molecular weight of 600 000 and appear in oriented specimens as flexible thin rods with a length around 240-250 run. The circular dichroism spectrum of the Ascaris protein is dominated by /J-structure. The proteins react with antibodies to insect mini-titin and also with antibodies raised against peptides contained in the sequence predicted for twitchin, the product of the Caenorhabditis elegans unc-22 gene. Antibodies to insect mini-titin decorate the body musculature as The giant protein titin is the major component of the elastic filaments of sarcomeric muscles from vertebrates (for reviews see Wang, 1985; Mamyama, 1986). Immunoelectron microscopy using a bank of different monoclonal antibodies has shown that the titin molecule extends from the Z-band into the M-band (Furst etol. 1988,1989). While the native titin molecule is still difficult to obtain, a defined proteolytic fragment TH, which spans the distance from the Ni line into the M line, is readily purified (Maruyama et al. 1984; Trinick et al. 1984; Wang et al. 1984; Nave et al. 1989). Til seems to be a single polypeptide of apparent molecular weight 2.1 xlO6 to 2.4X106 (Kurzban and Wang, 1988; Nave et al. 1989). When oriented on mica by centrifugal force, subsequent metal shadowing demonstrates uniformly thin rods with a diameter of 3-4 run and a length around 900 run (Nave et al. 1989). Thus the parent titin TI probably has the same length as a half-sarcomere. When flight and leg muscles of Locusta migratoria and other insects are subjected to a titin purification scheme, a protein of apparent molecular weight 0.6 x 106 is obtained. It resembles vertebrate titin in many physical-chemical properties, but has a length of only 260 run. In line with its reduced length, immunoelectron microscopy with rabbit antibodies locates the mini-titin at the I-band and the adjacent part of the A-band. Western blots and immunofluorescence microscopy show that mini-titin is present in the muscles of various invertebrates, including the well as the pharynx of wild-type C. elegans in immunofluorescence microscopy. In the twitchin mutant E66 only the pharynx is decorated. We conclude that the mini-titins of invertebrate muscles defined earlier by ultrastructural criteria are very likely to be twitchins, i.e. molecules necessary for normal muscle contraction. We discuss the molecular properties of the proteins in the light of the sequence established for twitchin. nematodes Ascaris lumbricoides and Caenorhabditis elegans (Nave and Weber, 1990). Recent cloning of the unc-22 gene of Caenorhabditis elegans predicts a body muscle protein of molecular weight 668520 built from repetitive domains related to the immunoglobulin superfamily. Mutants lacking the protein, which has been called twitchin, do not develop normal body muscle contraction and small regions of the myofilament organization in individual cells contract transiently in the absence of contraction in adjacent regions (Benian etal. 1989). In addition, a sizeable fraction of rabbit titin has been established by cDNA cloning (Labeit et al. 1990). The two cloning studies establish that twitchin and titin belong to the expanding family of proteins that covers the immunoglobulins, cell adhesion molecules and other muscle proteins such as C-protein, the 86K protein (Einheber and Fischman, 1990), and myosin light-chain kinase (Olson et al. 1990). Such molecules are built from repetitive 100-residue domains of two distinct types, designated motifs I and II. Among the collection of proteins in this superfamily, titin and twitchin show enhanced sequence homology. To relate the insect mini-titins characterized by their ultrastructure and their counterparts denned immunologically in nematodes (Nave and Weber, 1990) with the twitchin molecule of C. elegans predicted by cDNA cloning (Benian et al. 1989), we have used purification schemes for titin and mini-titin (Nave et al. 1989; Nave and Weber, 1990) on the nematodes C. elegans and Ascaris. We obtain flexible and thin rods with a length of around 245 run, which react with antibodies to insect mini-titin as well as with antibodies raised against two peptides from the predicted twitchin sequence. The combined results suggest that the mini-titins of invertebrates defined ultrastructurally correspond to the twitching defined functionally. Materials and methods Nematodes Ascaris was obtained from pigs at the local slaughterhouse. Wildtype C. elegans (strain N2) and the unc-22-deficient mutants (strain E66) were kindly provided by R. Schnabel (Max Planck Institute for Developmental Biology, Tubingen, FRG) and grown as described (Brenner, 1974). Body musculature of Ascaris was dissected. Tissue homogenization, washing of myofibrils and subsequent extraction steps were the same as those used for the isolation of minititin from Locusta migratona flight muscle (Nave and Weber, 1990). The final extract was dialyzed extensively at 4C against buffer T (50 mM Tris-HCl, pH7.9, 2mM EGTA, lmM 2-mercaptoethanol, lmM NaN3) containing 70 mM KC1 and clarified by centrifugation (100000g, l h ) . The supernatant was applied to a column (1.6cmx5cm for 5g tissue) of Q-Sepharose (fast flow, Pharmacia LKB, Uppsala, Sweden) equilibrated in buffer T plus 70 mM KC1. The column was washed with several volumes of the same buffer, and then with buffer T containing 150 mM KC1. Fractions containing mini-titin were pooled, dialyzed against buffer T containing 500 mM KC1 and subjected to high-resolution gel permeation chromatography (GPC) using a TSK 6000 PW column (7.5mmx600mm, LKB) equilibrated with the same buffer. The column was developed at room temperature at a flow rate of 12mlh~1. Fractions containing pure mini-titin were pooled. The same procedure was also used to isolate mini-titin from the small nematode C. elegans. Animals ( l g kindly provided by Dr Schnabel) were homogenized in toto in liquid nitrogen using a pestle and mortar. Subsequent steps were as above. Rabbit antibodies to mini-titin from Locusta migraioria have been described (Nave and Weber, 1990). Rabbit antibodies to two different peptides in the published sequence of twitchin (Benian et al. 1989) were also raised. The peptides were synthesized and purified by HPLC. They were conjugated to ovalbumin (chicken egg, Sigma A-5503) via an N-terminal cysteine with sulfo-mmaleimidobenzoyl-/V-hydroxy8ulfosuccinimide ester (sulfo-MBS, Pierce no. 22312) as crosslinker (Liu et al. 1979). The modifi (...truncated)