Immunological Properties and Cdna Sequence Analysis of an Intermediate-ﬁlament-Like Protein from Squid Neuronal Tissue

Journal of Cell Science 106, 1283-1290 (1993) 1283 Printed in Great Britain © The Company of Biologists Limited 1993 Immunological properties and cDNA sequence analysis of an intermediate-filament-like protein from squid neuronal tissue James Adjaye*, Philip J. Marsh and Peter A. M. Eagles† Department of Molecular Biology and Biophysics, The Randall Institute, King’s College London, 26-29 Drury Lane, London WC2B 5RL, UK *Present address: Max-Planck-Institute for Biophysical Chemistry, Department of Biochemistry, PO Box 2841, D-3400, Goettingen, FRG †Author for correspondence SUMMARY A cDNA library has been constructed in the expression (IF) proteins. The rod has the classical heptad repeats vector gt11 from mRNA isolated from squid (Loligo indicating coiled-coil-forming ability, and the predicted forbesi) optic lobes. The library was screened with anti- lengths of the coils are similar to coils 1a, 1b and 2 of bodies generated against purified squid neurofilaments. intermediate filaments. At the C-terminal end of the rod A positive clone was isolated, which harboured a gt11 there is a strongly conserved IF epitope, and a fusion recombinant having an insert size of 3.5 kb. Hybridiz- protein containing SNLK is recognised by the pan- ation analysis by Southern and northern blotting specific intermediate filament antibody, IFA. A poly- showed that the corresponding protein is encoded by a clonal antibody raised against SNLK has been used to single gene that gives rise to a transcript of 2.6 kb. show that the protein is present only in neuronal tissues Translation of the full nucleotide sequence of the gene and that it is immunologically related to neurofilaments revealed an open reading frame covering 557 amino from Myxicola but not from mammals. acids. This squid-neurofilament-like protein, SNLK, bears the characteristic N-terminal head, rod and C-ter- Key words: intermediate filaments, sequence analysis, squid minal tail domains present in all intermediate filament nerves INTRODUCTION Sequence information obtained from the intermediate filament proteins of invertebrates shows that they are related Intermediate filament proteins are expressed co-ordinately more closely to nuclear lamins than to their vertebrate coun- during development and cell differentiation. They are prob- terparts. For example the nuclear lamins of both vertebrates ably involved in special functions related to the differenti- and invertebrates have six extra heptads at the ends of coil ated state of cells such as the maintenance of resistance to 1b and a similar extension to this region has been found in mechanical stress. Some cells lack intermediate filaments, epithelial intermediate filaments from Helix pomatia so they do not perform essential ‘house keeping’ functions (Weber et al., 1988), and cytoplasmic intermediate fila- (Hedberg and Chen, 1986; Bartnik and Weber, 1989). ments from Ascaris lumbricoides (Weber et al., 1989). Intermediate filaments in the cytoplasm of vertebrate Lamin-like tail domains are also found in these invertebrate cells are composed of different but related polypeptides. intermediate filament proteins. Five subgroups for these proteins have been distinguished: The pattern of similarities is shared by neuronal inter- type I and II keratins; type III proteins, i.e. vimentin, mediate filaments as well. The two low molecular mass neu- desmin, GFAP and peripherin; the neurofilament proteins rofilament proteins (60 kDa and 70 kDa) from the squid, (NFL, NFM, NFH and a -internexin), which constitute the Loligo pealei, which have had their sequences deduced type IV proteins; and the nuclear lamins, which form the from the cDNA sequences, show the basic features of the type V. All these filamentous proteins share basic structural group of invertebrate intermediate filament proteins (Szaro principles, which are a central a -helical rod region that is et al., 1991). The squid neurofilament proteins, like all the flanked by N- and C-domains that are essentially non-heli- vertebrate neurofilaments (Eagles et al., 1990), have long cal (Steinert and Roop, 1988). The rod regions form coiled C-terminal extensions to their polypeptides, and this region coils - the structural building blocks of the filament back- has clusters of charged residues. bone. The nuclear lamins follow similar principles for their We describe in this paper a new neuronal protein, the construction and are thought to be related through evolu- cDNA sequence of which was obtained from a squid optic tion to the intermediate filament family of proteins (Weber lobe cDNA library. The protein is related to the interme- et al., 1989; Döring and Stick, 1990). diate filament class of proteins, yet it is quite distinct from 1284 J. Adjaye, P. J. Marsh and P. A. M. Eagles the squid neurofilament sequences so far examined. We polypeptides and a number of proteins in the region around a rel- have called this protein SNLK (squid neurofilament-like). ative molecular mass of 60 kDa. The polyclonal serum was freed of any E. coli reacting antibodies by immunoprecipitation with E. coli lysates. For the preparation of anti-SNLK, source gels of the MATERIALS AND METHODS fusion protein b-gal-SNLK were run and the region of the gel containing the protein removed. The gel was macerated in adjuvant and then injected. Antigens were injected together with Freund’s Preparation and screening of the library complete adjuvant followed by one or two booster injections in The Amersham (UK) cDNA synthesis and cloning system kit incomplete adjuvant. b-galactosidase-reacting antibodies were (l gt11) was used for the construction of the library. Total RNA removed from the serum by immunoprecipitation. IFA (Pruss et was isolated from squid (Loligo forbesi) optic lobes using guani- al., 1981) was obtained from the ascites fluid of cell lines express- dinium isothiocyanate as described by Kaplan et al. (1979). ing this antibody. Poly(A)+ RNA was obtained from the total RNA by oligo(dT) cellulose chromatography (Aviv and Leder, 1972). Double-stranded Preparation of DNA and RNA for hybridization cDNA with EcoRI linkers was prepared by the procedure of analysis Gubler and Hoffman (1983) and inserted into the unique EcoRI Genomic DNA from squid optic lobes was prepared as described site of l gt11 (Huynh et al., 1985); it was then packaged. The total by Jeffreys et al., 1977. DNA (5 mg) was digested with restric- packaged library, before amplification, contained 2.8´ 108 pfu/ml. tion enzymes then electrophoresed on a 0.8% agarose gel at 90 Plaques were screened for the presence of neurofilament epitopes V. Lambda DNA cut with HindIII was used as markers. Elec- essentially as described by Young and Davis (1983). Plaque lifts trophoresis of RNA (10 mg) was carried out on a 0.5% on nitrocellulose were screened with antibodies generated against agarose/formaldehyde gel. Transfer of DNA and RNA onto nitro- purified squid neurofilaments that had been previously dephos- cellulose membranes was as described (Southern, 1975; Thomas, phorylated. 1980). Filters were prehybridized at 42°C for 3 hours in 50% Sequencing and analysis of the 3.5 kb cDNA deionised formamide, 5´ SSC, 0.2% SDS, 5´ Denhardt’s solution and 100 mg/ml denatured salmon sperm DNA. For hybridizations, The 3.5 kb cDNA was subcloned into pUC19, and a set of uni- the 3.5 kb cDNA was labelled with [32P]dCTP (Amersham, UK) directional nested deletions was generated using exonuclease III by the oligonucleotide-priming procedure of Feinberg and Vogel- then mung bean nuclease (Stratagene, UK) as described by stein (1984). Hybridizations were carried out overnight at 42°C. Henikoff (1984). Double-stranded sequencing was carried out Filters were washed at a final stringency of 0.1´ SSC/0.1% SDS using the dideoxy chain-termination method (Sanger et al., 1977). at 65°C. Amersham Hyperfilm was used for autoradiography. The sequence was analysed with computer programs implemented within the University of Wisconsin Genetics Computer Group Immunocytochemistry (GCG) software on the VAX (Devereaux et al., 1984). Giant axons were dissected out intact from fresh squid, embed- Preparation of gt11 fusion proteins ded in paraffin wax then snap-frozen in liquid nitrogen. Sections (10 mm thick) were cut on a cryostat maintained at a temperature The clone harbouring the 3.5 kb cDNA was used to infect the of - 20°C. Air-dried sections were fixed with 95% ethanol/5% lysogenic strain E. coli Y1089 as described by Huynh et al. (1985). acetic acid for 10 minutes at - 10°C. Sections were blocked in 2% Fusion proteins were produced by induction with 10 mM IPTG. non-fat milk in TBS for 1 hour, washed several times in TBS and Bacterial pellets were boiled in sample buffer (1% SDS, 100 mM exposed for 1 hour at room temperature to anti-SNLK (1:1000 Tris-HC1, pH 6.8) and spun briefly. The proteins were resolved dilution). Sections were then washed with TBS and further incu- by SDS-PAGE on a 5-15% gel (Laemmli, 1970) then either bated for 1 hour with rhodamine-labelled goat anti-rabbit anti- stained with Coomassie Blue or electrophoretically transferred bodies (Amersham, UK) diluted 1:50 in blocking solution. After onto nitrocellulose (Towbin et al., 1979). further washes, the sections were layered with an antiphoto- bleaching agent (ascorbic acid, pH 7.0, 5 mg/ml), covered with a Immunoblotting coverslip, then viewed using fluorescence microscopy and pho- Nitrocellulose filters containing proteins were blocked for 1 hour tographed. with 2% (w/w) non-fat milk with TBS (150 mM NaC1, 200 mM Tris-HCl, pH 7) then incubated with primary antibody for 1 hour at room temperature. After three washes with TBS/0.2% Tween- RESULTS 20, peroxidase-conjugated donkey anti-rabbit antibodies were added for polyclonal antibodies or anti-mouse for monoclonal antibodies (Amersham, UK) diluted 1:1000 in 2% non-fat milk Screening of the gt11 library and further incubations carried out for 1 hour.

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