Differentiation (1992) 52:33-43 Ontogcoy, Neoplasia and Differentiation Therapy

0 Springer-Verlag 1992

Cloning of a type I keratin from goldfish optic nerve: differential expression of keratins during regeneration Robert K. Druger', Edward M. Levine', Eric Glasgow', Paul S. Jones2, and Nisson Schechter'.2

' Department of Biochemistry and Cell Biology, Health Sciences Center, State University of New York, Stony Brook. New York 11794, USA * Departrncnt of Psychiatry and Behavioural Science, Health Sciences Center, State University of New York, Stony Brook, Ncw York 11794, USA Accepted in revised form 20 August 1992

Abstract. We report the cDNA sequence and predicted amino acid sequence of a novel type 1 keratin, designated as GK50, and show that keratin expression in the goldfish optic nerve is highly complex. The GK50 protein is one of at least three type I keratins expressed in goldfish optic nerve based on both antibody reactivity and blot-binding to the type 11 keratin ON,. After optic nerve crush in situ hybridization shows a localized increase in GK50 mRNA expression in the crush zone. This is in contrast to ON, mRNA which shows a localized increase that is limited to the proximal and distal margins of the crush zone, suggesting a diversity of keratin expression in different cell types of the goldfish optic nerve.

Key words: Glials cells - Intermediate filaments - Keratins - Optic nerve - Regeneration

Introduction Microfilaments, microtubules and intermediate filaments are distinct filamentous networks that comprise the cytoskeleton of most eukaryotic cells. The intermediate filament proteins, in contrast to the proteins of the two other cytoskeletal networks, are structurally diverse [45]. In addition, these proteins are expressed in a cell specific manner and their expression is developmentally regulated [15]. Although a precise function has not been assigned to this class of proteins, their structural heterogeneity and their distinctive pattern of expression indicates that particular intermediate filament proteins may have structural attributes that meet physiological requirements of the differentiated state of the cell. Intermediate filament proteins have been categorized into six subclasses according to their cellular deposition and primary structure [62]. The type I and type I1 keratins (acidic and basic) are expressed in epithelial cells [42]. The type I11 intermediate filament proteins include Correspondence lo: N. Schechter

vimentin in mesenchymal cells, desmin in smooth muscle, glial fibrillary acidic protein (GFAP) in astrocytes and peripherin which is abundant in peripheral nerves and some CNS neurons [47, 481. Type IV intermediate filament proteins consist of the neurofilament triplet NF-L, NF-M and NF-H, which are localized to neurons [32,62], and a-internexin, which is specific for embryonic CNS neurons [13]. The type V intermediate filament proteins are the nuclear lamins, which are part of the karyoskeleton [52]. Finally, nestin is a type VI intermediate filament protein that is localized to CNS stem cells I281. The expression of intermediate filament proteins is closely linked to the developmental state of a tissue. There is a regulated expression of intermediate filament proteins during development and their expression is often used as a marker for differentiation. This is especially true for keratins [14, 151. Of all the different types of intermediate filament proteins, the keratins represent the most complex group with over 20 different polypeptides identified [42, 451. Type I and type I1 keratins are selectively expressed as pairs and, unlike other types of intermediate filament proteins, form obligate heteropolymers when forming filaments in vitro [21] and in vivo [20]. The expression of a specific keratin pair is regarded as a marker for different epithelial cell types and for epidermal differentiation [17]. Keratin expression is not limited to epithelial tissue. Keratins are found in oocytes and spermatozoa of several species [7, 591 and are among the first differentially expressed gene products during embryogenesis [46]. In addition, keratins are expressed in several non-epithelial tissues of adult lower vertebrates [38, 391. It is noteworthy that optic nerves of lower vertebrates maintain a capacity for continued growth and development [12,23]. Furthermore, the predominant glial intermediate filament proteins in the optic nerves of lower vertebrates are keratins [18, 19, 561 and not the expected GFAP, which is the intermediate filament protein expressed in glial cells of higher vertebrates [9]. The unexpected expression of keratins in glial cells of lower vertebrate optic

34

nerves may impart structural attributes which fulfill physiological requirements of these systems [18, 19, 35, 581. An important example is the goldfish visual pathway [18, 191. The goldfish visual pathway displays continuous growth, development and plasticity throughout life [I 2, 23, 411. Furthermore, a remarkable capacity for functional regeneration occurs after optic nerve injury [60]. The intermediate filament proteins of the goldfish optic nerve do not match the conventional intermediate filament composition of the more static postdevelopment mammalian optic nerve [54]. The glial protein ON, was previously cloned by this laboratory and was found to be a type I1 keratin structurally similar to human keratin 8 [18]. The expression of this type I1 keratin predicts the expression of a type I keratin partner, a requirement for filament formation [20,63]. Furthermore, since keratins are expressed in a cell specific manner, the recent identification of several non-neuronal cell types in the goldfish optic nerve [I I ] suggests the expression of additional keratin pairs. The present study was undertaken to examine the complexity of keratin expression in the goldfish optic nerve. Using a polymerase chain reaction (PCR) protocol we have isolated and sequenced the complete cDNA of a goldfish optic nerve type I keratin, designated GK50 (Goldfish Keratin; 50 kDa). The GK50 protein is one of at least three type I keratins in goldfish optic nerve, demonstrating a complex pattern of keratin expression in this tissue. During optic nerve regeneration there is a localized increase in expression of GK50 mRNA which is limited to the crush zone. This contrasts with the type I1 keratin ON, mRNA which increases at the proximal and distal margins of the crush zone.

Methods Animals. Common goldfish (Carassius auratus) were obtained from Mt. Parnell Fisheries (Mercersburg, Pa., USA) and maintained in 40 gallon tanks at approximately 18" C. Intraorbital nerve crush was performed on the right optic nerve after anesthetization by immersion in 0.05% tricaine methanesulfonate. The left optic nerve was left intact to serve as a control. Cytoskeletal preparation and two-dimensional electrophoresis. Cytoskeletal proteins were isolated from goldfish optic nerve as described previously [26]. Two dimensional electrophoresis was performed essentially as described by O'Farrell[44] with slight modifications [53]. Autoradiography was performed as described previously [191. RNA isolation. Tissue was immediately frozen in liquid nitrogen and total cellular RNA was isolated with RNasol B according to the manufacturers instructions (Tel-Test). Poly A + RNA was selected from total RNA by passage over a column of oligo(dT)cellulose [3].

In vitro transcription and translation. RNA transcripts of the GK50 and ON3 cDNAs were synthesized in vitro using the T3 and T7 promoters after linearization of the phagemid pBluescript KS(Stratagene) containing the 1.6 kb (GK50) or 2.2 kb (ON,) insert. Optic nerve poly A+ and transcribed RNA from both GK50 and ON, cDNAs were translated in a rabbit reticulocyte lysate contain-

ing [ 35S]-methionine according to manufacturers instruction (Promega). An aliquot from each translation reaction was mixed with 30 pg of a goldfish optic nerve cytoskeletal preparation and analyzed by both one and two-dimensional polyacrylamide gel electrophoresis. Five microlitres from the translation reactions containing approximately lo5 trichloroacetic acid (TCA) precipitable counts were used for blot-binding assays. lmmunoblotting and blot-binding assay. After separation by twodimensional electrophoresis, 30 pg of goldfish cytoskeletal protein was transferred to either nitrocellulose (Schleicher and Schuell; for immunoblot) or PVDF (Millipore; for blot-binding) membranes using the method of Towbin et al. [64]. Immunoblots were performed as described previously [ 191. Blots were incubated with primary antibody AE1 (Amersham) at a 1 :2000 dilution. AE1 is a monoclonal antibody which recognizes most type I keratins [63]. Secondary antibody was alkaline phosphatase-conjugated (Sigma). Blots were developed in NBT/BCIP according to manufacturers instructions (Bethesda Research Labs). For the blot-binding assay, PVDF blots were first visualized by staining with amido-black (0.025"/, in 45% ethanol, 10% acetic acid) and destaining (45% ethanol, 10% acetic acid). Blots were then blocked in TBST (20 mM TRIS-HCI (pH 7.4), 150 mM NaCl and 0.05% Tween-20) containing 5% non-fat dried milk for 30 min. After several washes with TBST, the blots were incubated in 10 ml TBST containing 5% non-fat dried milk and approximately lo6 TCA precipitable counts of either translated ON, or GK50 directly from in vitro translation reactions and allowed to shake at 20" C for 1 h. After washing in TBST several times, the blot was air dried and exposed to X-ray film (Kodak XAR-5). Immunoprecipitation. Samples were immunoprecipitated directly from translation reactions. Five microlitres containing approximately lo6 cpm of TCA precipitable protein was added to 200 pl of immunoprecipitation (IP) buffer (20 mM TRIS-HCI (pH 7.4), 100 mMNaCl,5 m M EDTA, 1% deoxycholate, 1% Triton X-100). One microlitre of undiluted AEl (Amersham) antibody was added and the reaction was incubated at 20" C for 2 h. Ten microlitres of undiluted rabbit anti-mouse immunoglobulin G (IgG) (Sigma) was added and the mix was incubated overnight at 4" C. The precipitate was then collected by centrifugation and washed three times in IP buffer prior to analysis by two-dimensional electrophoresis. Screening of the cDNA library. Construction of a goldfish optic nerve library was carried out as described previously [18]. Initial screening of the library utilized the polymerase chain reaction (PCR). Primers were made that correspond to conserved regions of intermediate filament proteins. A degenerate 5' primer (5'-GAT/ CAAT/CGAAGCTTGGCXGCXGAT/CGAT/CTT-3') was made which represented the amino acids DNARLAADDF. This sequence is highly conserved among type I acidic keratins. A Hind 111 restriction site was incorporated into the primer to facilitate subcloning. A 3' primer (5'AGAAGCTTCCTGTATGTGGCGATCTC-3') was made to the sequence EIATYRRL which is highly conserved in all intermediate filaments. This primer was not degenerate as the sequence for ON, [ 181 was used to generate the sequence. A single base change incorporated a Hind 111 site into this primer. Initially a PCR reaction was run with lgtl 1 forward and reverse primers (New England Biolabs) using the cDNA library as template to amplify the cDNA inserts. The PCR reaction consisted of: 1.6 mM MgCI2, 10 mM TRIS-HCI (pH 8.3) , 50 mM KCI, 200 F M dNTPs, 250 ng of each primer, and 2.5 units Taq polymerase (Stratagene) in a 100 p1 volume. One microlitre amplified optic nerve cDNA library was used as a template. Thirty PCR cycles were performed with denaturing at 94" C for 30 s, annealing at 50" C for 2 min and extension at 72" C for 3 min. A 1 p1 aliquot from this reaction was used as template for a second PCR reaction using the type I keratin specific and intermediate filament specific primers. The previous PCR reaction conditions were used.

35 The reaction was then analyzed on a 0.8% agarose gel and a 700 base-pair band was observed. This DNA band was purified from the gel (Geneclean kit, BIO101) and digested into fragments with Taq I restriction endonuclease, since subcloning using restriction sites incorporated into the primers proved to be problematic. These were subcloned into phagemid pBluescript KS- and sequenced [57]. An 82 base-pair fragment whose sequence corresponded to a core region from a type I keratin was used to rescreen the l g t l l cDNA library in order to isolate full length clones. The fragment was purified from a 2% agarose gel (Mermaid kit, BIOlO1) and labelled with [3ZP]-dCTP (New England Nuclear) by random primer synthesis (Amersham). This was then used to screen the library at high stringency for full length clones as described in Current Protocols in Molecular Biology [2]. Nine clones containing inserts with identical restriction patterns were isolated. The largest clone (1.6 kb) was subcloned into phagemid pBluescript KS- and the clone sequenced [57] fully in both orientations utilizing subcloned restriction fragments and synthesized primers. The nucleotide sequence reported here has been submitted to the GenBank Data Bank and the assigned accession number is M86918. Northern-blot analysis. Five micrograms of total RNA from optic nerve, retina, brain and spinal cord was resolved on a 1.3% agarose/2.2 M formaldehyde gel in 1 x MOPS (20 mM 4-morpholinopropanesulfonic acid (pH 7.0), 5 m M Na acetate and 1 mM EDTA). The RNA was transferred onto Nytran (Schleicher and Schuell) according to the manufacturer's instructions. The blot was prehybridized for 1 h, and hybridized overnight at 42" C in 1 x PE (50 mM TRIS-HCI (pH 7.5),0.1% Na pyrophosphate, 1% sodium dodecyl sulfate (SDS), 1 x Denhardts reagent and 1 m M EDTA), 5xSSPE (0.9M NaCI, 50mM Na,PO, (pH 7.7) and 5 m M EDTA), 50% formamide, 0.1 mg/ml calf thymus DNA, and 10 pg/ ml yeast tRNA. The entire GK5O cDNA was labelled with [,'PIdCTP by random primer synthesis and added to the hybridization mix at a concentration of lo6 cpm/ml. After hybridization, the blot was washed in 2 x SSC (0.3 M NaCI, 30 m M Na citrate, pH 7.0). containing 0.1 TOSDS. The final wash was at 55" C for 45 rnin and the filter was autoradiographed with Kodak XAR-5 film with an intensifying screen at - 80" C for 4 days. In situ hybridizations. Goldfish were anesthetized in ice prior to tissue removal. To fix the tissue, the optic nerves were immersed in 4% paraformaldehyde in RNase-free PBS (0.1 M sodium phosphate, pH 7.4, 0.9% NaCI) for 2 h. Crushed and control nerves were embedded together in a 1 :1 mixture of OCT embedding medium (Miles Labs) and Aquamount (Lerner Labs) as previously described [25]. Cryostat sections were cut at 8 microns and collected on to aminoalkylsilane treated slides [ 551. Slides were frozen at -80" C and lyophilized for 30 min. They were then warmed to 45" C and washed with three 5 min changes of RNase-free PBS, followed by a 10min wash in 0.25% acetic anhydride in 0.1 M triethanolamine buffer (pH 8.0) followed by 2 washes in RNase-free PBS. All washes were at 20" C. Prehybridization and hybridization were carried out in the same buffer: 50% deionized formamide, 600 m M NaCl, 10 mM TRISHCl (pH 7.5). 1 m M EDTA, 0.02% Ficoll, 0.02% polyvinyl pyrollidone, 1 mg/ml bovine serum albumin (BSA), 25 m M dithiothreitol (DTT), 0.02% SDS, 1 mg/ml tRNA, and 1 mg/ml salmon sperm DNA. The slides were prehybridized at 39" C for 1 h. GK5O cDNA probes were labelled with [35S]-dCTP by random priming (Pharmacia). These probes were denatured by heating at 95" C for 5 min before mixing with the hybridization buffer. Each slide was incubated at 39" C overnight in 100 pl hybridization buffer containing approximately lo6 cpm of probe. After incubation, the slides were washed two times in 4 x SSC (0.6 M NaCI, 60 mM NaCitrate, pH 7.0) and 1 mM DTT at room temp for 10 min, one time in 4 x SSC at room temp for I0 min, once in 2 x SSC (0.3 M NaCI, 30 mM NaCitrate, pH 7.0) at 39" C for 30 min and two times in 0.2 x SSC (0.03 M NaCI, 3 m M NaCitrate, pH 7.0) at 39" C for 30 min. The slides were then air dried,

dipped in a 1 :1 dilution of Kodak NTB2 emulsion, air dried again and allowed to expose for approximately one week at 4" C. The dipped slides were developed in a 1 :1 solution of Kodak D-19 developer at 18" C for 4 min, followed by a 5 rnin wash in Kodak fixer and air dried. The slides were then examined using dark field microscopy. RNase protection assays. RNase protection assays were performed essentially as described by Melton et al. [40]. A 326 base-pair EcoR 1-Nar 1 fragment, representing nucleotides 1 to 326 of the GK50 cDNA, was subcloned into phagemid pBluescript KS-. This construct was linearized with EcoR 1 and used to synthesize a 373 nucleotide GK5O antisense riboprobe by in vitro transcription with T3 polymerase in the presence of [32P]-UTP. A 118 base-pair Sau3A fragment, representing nucleotides 750-868 of the ON, cDNA, was subcloned into phagemid pBluescript SK (Stratagene). This construct was linearized with EcoR l and used to synthesize a 183 nucleotide ON, antisense riboprobe as above. The labelled riboprobe was purified on an 5% polyacrylamide gel. Total RNA (20 pg) from both 10 day post crush and uncrushed optic nerve was hybridized with 5 x lo5 cpm probe at 45" C overnight. Single-stranded RNA was digested with RNase A (4 pg/ml) for 1 h at 37" C. The protected RNA was analyzed 011 a 6% sequencing gel.

Results Cytoskeletal proteins expressed in the goldfish optic nerve

The major intermediate filament proteins in the goldfish optic nerve are a group of proteins (Fig. 1 A) at 58 kDa and a protein at 48 kDa [52]. The 58 kDa proteins can be separated into at least four isoelectric variants and are designated as ON,-ON, [50]. Proteins ON, and ON2 are of neuronal origin whereas the nonneuronal intermediate filament proteins ON3 and ON, and the 48 kDa protein are of glial origin [51]. Several less abundant proteins, including a protein at 50 kDa denoted as GK50 and a basic protein (PI 6.8) at 48 kDa [52], are also observed. Proteins ON3 and ON., are similar in structure and ON, may be a phosphorylated form of ON3 [52]. The ON3 protein was previously cloned by this laboratory and was found to be a type I1 keratin

Expression of type I keratins in the goldfsh optic nerve

A monoclonal antibody against human type I keratin (AE1) [63] was used to characterize intermediate filament proteins expressed in the goldfish optic nerve by Western blot analysis (Fig. 1 B). The antibody reacted with the GK50 protein, an isoelectric variant of GK50, and the 48 kDa and its isoelectric variants. The AE3 antibody also reacted with a basic protein (PI 6.8) at 48 kDa found in the optic nerve [52]. Type I und I1 keratins form obligate heteropolymers when forming filaments and show specific binding with each other in vivo [20] and in vitro [22, 331. Although the expression of specific keratin pairs occurs in vivo, there is a relaxation of specificity in vitro and almost any type I keratin will bind with any type I1 keratin, even across species lines [21]. This binding affinity was utilized to further characterize the type I keratins ex-

36

Characterization of the GK50 cDNA clone and classification as a type I keratin

Fig. 1. Distribution of type I keratins in the goldfish optic nerve. A Two-dimensional gel of cytoskeletal proteins isolated from the goldfish optic nerve. Brackers denote the ON proteins (ON,ON,) and the 48 kDa protein and its isoelectric variants. The GK50 protein and an isoelectric variant is brucketed. A carer marks the position of a basic protein at 48 kDa found in the nerve [52]. The positions of tubulin ( t ) and actin (a) are marked for reference. B Western blot of goldfish optic nerve cytoskeletal proteins probed with antibody AEl showing type I keratins by antibody reactivity. C Blot-binding assay using in vitro transcribed and translated ON3 protein as a probe showing type 1 keratins by affinity for the type I1 keratin ON,

pressed in the goldfish optic nerve. When a two-dimensional gel of a goldfish optic nerve cytoskeletal preparation was blotted to a membrane and then probed with labelled ON,, the type I1 keratin ON, showed affinity to the same proteins that reacted to antibody AEI (Fig. IC). The GK50 protein, the 48 kDa protein and the basic protein at 48 kDa all demonstrated specific binding with the type I1 keratin ON,.

Using a PCR protocol, a full length cDNA clone coding for the GK5O protein was isolated from a goldfish optic nerve Agtll library (see Methods). The nucleotide sequence is 1521 nucleotides long and includes a 31 basepair 5' noncoding region and an 89 base-pair 3' noncoding tail (Fig. 2). The noncoding tail includes a polyadenylation site followed by a short stretch of poly-A. The predicted amino acid sequence of the cDNA is 467 amino acids long. The predicted molecular weight of the GK50 protein is 49,757 daltons, in agreement with an estimated molecular weight of 50 kDa. Immunoprecipitation of in vitro transcribed and translated GK50 cDNA with the type I keratin specific AE1 antibody [63] confirmed the identity of the cDNA as coding for the GK50 protein (Fig. 3). The AEI antibody was first used to immunoprecipitate in vitro translated optic nerve poly A + RNA (Fig. 3A). The major immunoprecipitated product was the GK50 protein and an isoelectric variant (Fig. 3B). A small amount of the 48 kDa protein was also immunoprecipitated. The reactivity of the AE1 antibody with the GK50 protein is more robust on a Western blot when compared to the 48 kDa protein (Fig. 1 B). This may explain why the 48 kDa protein did not immunoprecipitate using this antibody as readily as the GK50 protein. In vitro transcribed and translated GK50 cDNA comigrated with the GK50 protein on a two-dimensional gel (Fig. 3C) and was also immunoprecipitated by the AEI antibody (Fig. 3D). The amino acid sequence displays a structure consistent with all intermediate filament proteins [62]. Structural analysis (using GCG; [lo]) of the 309 amino acid central core is in agreement with intermediate filament protein conformation with three coiled-coil domains (coil lA, lB, and 2A) interrupted by two nonhelical linker sequences (LI, L2). The intermediate filament protein consensus sequence TYR(X)LLEG [29] is at the end of coil 2 with one amino acid modification in which threonine (T) is replaced by glutamic acid (E). The sequence has several characteristics which classifies it as a type I keratin (Fig. 4). The 115 amino acid N-terminal domain displays features common to all keratins, such as several glycine-rich repeats. In addition, the C-terminal domain is rich in serine, threonine and valine. This is similar to the amino acids found in this domain of other keratins [24]. Also, the sequence DGKVVS in the C-terminal domain is found in many other type I keratin intermediate filament protein sequences [66]. Finally, the GK50 protein contains the sequence DNAKLATDDF in coil 1B. This is very similar to the sequence DNA(R/K)LAADDF which is found in many type I keratins [24, 29, 37, 661. When compared for structural similarity to other known keratin sequences, the GK50 amino acid sequence is similar to keratin 14 [37] and keratin 15 [29] in the human keratin catalogue and to developmentally regulated keratins expressed in Xenopus laevis embryos, XK70 [66] and XK81 [24]. The percentage of amino

37 1 1

15 76

H T S F S R Q S F H S S G G G ATG ACG TCC TTT AGC CGC CAG AGT TTC ATG TCT TCT GGA GGG GGT

5'-GCAMMGACCATCTCCTCTGCACCCCAGCA

77 ATA GGT GGA AGC TCC ATG CGT GGT CCT TCT ATG GGA AGC ATG TCT CGG TCA TCT GGG A T 1 GGT GGT GGA GGC GGG GGC

41 154

H I S A H R A G S V Y G G A G G H G V R I S T G T R 42 155 CAT ATA AGC GCC ATG CGT GCT GGC ACT GTG TAT GGG GGT GCA GGA GGC CAC GGG GTC CGT ATC TCC ACT GGG ACA AGA

67 232

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T F A S G G G G G G G A S Y G F G G G S G G G G G F 233 ACC T T T GCA TCT GGT GGT GGA GGC GGC GGT GGA GCC AGC TAT GGC TTT GGT GGT GGC TCT GGT GGC GGA GGA GGG TTT

93 310

G Y G S G A G G G F G G G D M D A K V N V S 94 311 GGC TAT GGT AGT GGC GCC GGA GGA GGC TTC GGA GGA GGC GAT ATG GAC GCC MA GTG M T GTC AGT

119 388

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544

I R D L P D H I P D A T R I N G G V Y L A I D Y A K L A 545 GAC CTG CM GAT ATG ATC CM GAT GCC ACT CGC ATC M T GGG GGC GTC TAC CTG GCC ATC GAT M C GCA AM TTG GCC

197 622

198 T D D F K T K Y E N E L A H R a s v E A D I A G L K 623 ACT GAC GAC TTC M G ACC M G TAT GAG M T GAG TTG GCC ATG CGT CM TCT GTG GAA GCA GAT ATC GCT GGG CTG M G

223 700

L D E L I L A R s D L E n a I E G L K E E L I Y 224 R L 701 AGA CTC CTT GAT GAG CTG ACA CTG GCA AGA TCC GAC CTG GAG ATG CAG ATT G M GGC TTA M G GAG GAG CTT ATC TAT

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276 857

A P Q E D L S R V H A E I R E P Y E G V S A K N Q R GCT CCT CAG GAG GAC CTG AGC CGC GTG ATG GCT GAG ATC CGA GAG CAG TAC GAG GGA GTC AGT GCC MA M C CAG CGG

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328 s K T E v T E L R R T L a G L E I E L a s E L s K K 353 1013 TCC MG ACT GM GTC ACT GAG CTT AGA CGC ACC CTT CM GGC CTG GAG ATC GAG TTA CM TCT GM CTC AGT MA MA 1090 R s L E G T L A D T E s R Y s I a L T a L a A R v T 354 1091 AGA TCT CTT GAG GGA ACT C T t GCA GAT ACA G M TCG CGT TAC TCC ATA CAG CTG ACT CAG CTA CAG GCT CGT GTA ACA

379 1168

u)o S L E E P I V H L R G D H D R Q S ~ E Y ~ H L L 1169 AGT CTG GAG GAG CAG ATC GTT U C CTC AGA GGT GAT ATG GAC AGA CAG TCT CAG GAG TAC MA ATG CTG TTG GAC ATC

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Fig. 2. The cDNA and predicted amino acid sequence of GKSO.The GKSO clone is 1521 nucleotides in length and codes for a full length protein of 467 amino acids. The conserved helical core region is shown in hackers. The polyadenylation signal AATAAA is in boldface

acids that are identical between GK50 and seven intermediate filament proteins, including three type I keratins, for different regions of sequences aligned for maximum structural identity are shown in Table 1. The highest region of similarity between the GK50 protein and other intermediate filament proteins is in the three helical regions HlA, and H1B and H2.

GK.50 expression in other nervous tissues ofthe goldfish Northern-blot analysis was performed on goldfish total RNA using GK50 cDNA as a probe to determine whether a full length sequence was represented and whether GK5O expression was limited to the optic nerve. This analysis reveals that the cDNA for GK5O represents a full length mRNA of 1.6 kb (Fig. 5). The expression of GK50 mRNA is not limited to the optic nerve, but is

N

38

A

also expressed at lower levels in both the spinal cord and brain of the goldfish, while no expression was observed in goldfish retina. Binding specificity of the GK50 protein to type II keratins in the goldfish optic nerve Since the GK50 protein is a type I keratin, it was expected to have a specific binding affinity for type I1 keratins in vitro [22]. A blot-binding assay was performed on goldfish optic nerve cytoskeletal proteins using [35S]-methioninelabelled GK5O protein as a probe. The GK50 protein was found to have a specific affinity for the ON, protein, its isoelectric variant ON, and possibly a third component localized between the two (Fig. 6). There was also an affinity for a 44 kDa protein (PI 5.4) and for several proteins of lesser abundance that migrate in the vicinity of ON, and ON,. These results demonstrate the distribution of type I1 keratins in the goldfish optic nerve based on binding affinity to the type I keratin GK50.

C

D

ON3 and GKSO mRNA levels during optic nerve regeneration

In situ hybridization experiments show that the pattern of mRNA expression of the type I1 keratin ON, is different from that of the type I keratin GK50 during optic nerve regeneration. In situ hybridization on uncrushed control nerves shows an even distribution of both GK50 and ON, mRNA throughout the nerve (data not shown). Ten days after optic nerve crush, mRNA levels for ON, increase at the proximal and distal margins of the crush zone (Fig. 7A), whereas the mRNA levels for the GK50 keratin increase in the crush zone (Fig. 7C). RNase protection assays show that there is no overall increase in the amount of ON, mRNA (Fig. 7B) and an overall decrease in mRNA for GK50 (Fig. 7D) 10 days after optic nerve crush. The mRNA for the protection assays was isolated from the entire optic nerve and therefore represents processes associated with regeneration as well as degeneration in this tissue. This may explain the apparent discrepancy between mRNA levels of the RNase protection experiments and the in situ hybridizations.

Discussion Fig. 3. A cDNA for the type I keratin codes for the GK50 protein. A In vitro translated goldfish optic nerve poly A' RNA was analyzed by two-dimensional electrophoresis. The GK50 protein is denoted by a bracket. The ON3 protein and the 48 kDa proteins are also designated by brackets. B Immunoprecipitation of GK50 from in vitro translated total poly A + RNA with AE1 antibody. C In vitro transcribed and translated cDNA clone of GKSO. D Immunoprecipitation of GK50 with the AE1 antibody after in vitro transcription and translation of a cDNA clone of GK5O

The goldfish visual pathway, unlike the visual pathway of higher vertebrates, retains continuous growth and development throughout life [12, 23, 411 and is capable of functional regeneration [60]. We report the sequence of a novel type I keratin, designated GK50. This protein shows a localized increase in mRNA in the crush zone during optic nerve regeneration. Furthermore, we demonstrate a complexity of keratins in the goldfish optic nerve.

39

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GK50 HK14 ......................................................MT-

MTSFSR TCS

GK50 QSFMSSGGGIGGSSMRQPSMGSMSRSSGIGGGQGGHIS~QSVYQGAGGHG~ISTGT HK14 RQFTSSSSMKQSCGIGGGIGAGSSRISSVLAGQSCRAPNTYGQGLSVSSSRFSSGGAYQL XK81 ..MTSYRSSSASYYSGSSSKG---GFGS ON3 MSYTKKTSYSVKSSSSGSVPRS-FSSMSYSQPSVTRQSYSVRTSYGGANRQMQAGMGGQG

................................

GK50 HK14 XK81 ON

RTFASQQQGGGGASYQFGGGSQGGGGFQYQSGAQGQFGGGDMDA~SE~TMQN~R GGGYGGGFSSSSSSFQSQFQGGYQQGLGAQMGOFOGaFAGGQFAGGDGLLVGSE~TMQN~R RSLA-GSNSYGGSSFGAGFSSQVQSGF--SSSGGNF~EAASSSFGGNE~QN~DR FISSSSAYGMMGMGSGWAPIQAVTFNKSLLAPLNLEIDPNIQWRTQEKEQMKSLNNR LGTMPRFSLSRMTPPLPARVDFSLAGALNAGFKETRASERAEMELNDR

GFAP ...........

GK50 WLTYLEKVHSLEKZLNGDLELKIRQFLENKTS----PDARDYSAYHATISDLQDMIQDATR HK14 LASYLDKVRALEEANADLEVKIRDWYQRQRP----AEIKDYSPYFKTIEDLRNKILTATV XK81 LASYLEKVRALEATNSDLEGKIRNWYDKQSDAGIGAGIGAGSKDYSKYFEIIAELRNKI~TI FASFIDKVRFLEQQNKMLETKWSLLQNQTAT----RSNID-AMFEAYINNLRRQLDSLGN E k P FASY IEKVRFLEOONKALAAELNOLRAKEPT------- KLADVYOAELRELRLRLDOLTA GK50 I N G G V Y t A F D N A K L A T D D F K T K Y g N E L A M R P s V E A D I A O L D L E M Q I E G HK14 DNANVLLQIDNARLAADDFRTKYETELNLRMSVEADINQLRRVLDELTLARADLEMQIES XK81 DNATVTLQIDNARLAADDFRLKFENEfiALRQBVEGDSNaLRRVLDELILARGDFELQIES DKMKLEADLHNMQGLVEDFKKYEDEINKRTECENDFVL1~DVT)EAYMNKVELEAKLES Z k P NSARLEVERDNFA0DU;TLROKLODETNLRLEAENNLAAYROEAHEATLARVDLERKVES GK50 HK14 XK81 ON

GFAP

LKEELIYLKKNHEEELASMRSQMTG~E-VDMPQEDLSR~IREQYEGVSA~QR LKEELAYLKKNHEEEMNALRGQVGQDVNVE-MDAAPGVDLSRILNEMRDQYEKMAEKNRK LTEELAYLKKNHEEEMSHAKSQSAGKVSVE-MDAALGVDLTSILNNMRADYEILAEKNRR LSDEINFLRQIFEEEIRELQSQIKDTSVWEMDNSRNLDMDAIVAEVRAQYEDIANRSRA

~EEEIQFLRKIYEEEVRDLREQ-LAQQQVKVEMDVAKPDLTAALREIRTOYEAVATSMNQ

GK50 ELDAWFQTKSETLTKEVTANTETLQVSKTEVTELRRTLQGLEIEVQSELSKKRSLEGTLA HK14 DAEEWFFTKTEELNREVATNSELVQSGKSEISELRRTMQNLEIELQSQLSMKASLENSLENSLE XK81 DAELWFNQKSGELKKEISVGVEQVQAS~EITELKRSLQSLEIELQSQLAMKQSVEGNLN

EAEMWYKSKYEEMQTSATKYGDDLRSTKTEIADLNRMIQRLQSEIDAVKGQRSNLENQIA Z k P ETEEWYRS KFADLTDAASRNAELLROAKHEANDYRROLOALTCDLEsLRGTNEsLERo~ GK50 DTESRYSIQLTQLQARVTSLEEQIVHLRGDMDRQSQEYQMLLDIKTRLEMEI~YRRLLD HK14 ETKGRYCMQLAQIQEMIGSVEEQLAQLRCEMEQQNQEYKILLDVKTRLEQEIATYRRLLE XK81 ELQGFYSSQLQQIQNTIGSLEEQLLQIRSDHEHQNTEYKLLLDIKTRLEMEIQTYRRLLE

EAEERGELAVRDAKARIKDLEDALQRAKQDMARQIREYQELMNVKLALDIEIATYRKLLE Z k P EOEERHARESASYOEALARLEEEGOSLKEEMARHLOEYODLLNVKLALDIEIATYRKLLE

****** G K ~ OOGA--TSFSTSGGGGQGGGGWSSTKTITV--KTIEEDIVDGKVVSST------ TK HK14 GED--AHLSSSQFSSGSQSSRDVTSSSRQI--RT~DVHDGKVVS-THEQVLRT~ XK81 GELGQVTTVANTSSVESKTESSSTSTTRTRMVKTIVEEWDGKVVSSRVE ON^ GEE--DRLLSGIKSVNISKQSTSYGSYPMESASSGYSNYSSGYGGYGGGGYSSGGGYSSG GFAP ~--NRITIPVQTFSNLQIRETSLDTKSVSEGHLKRNI----WK-TVEMRDGEVIKDS ON3

GGYSSGGGYSSGSGYSETVSQTKKSWIKMIETKDGRWSESSEWQD

GFAP KQEHKDVVM Table 1. Percent amino acid identity between the GK50 protein and other intermediate filament Droteins

H1 HK15 HK14 XK70 XK81 HK18 ON3 GFAP

37 28 13 26 14 16 2

Helix

Fig. 4. Comparison of amino acid sequence of-GK5O to other keratins and glial fibrillary acidic protein. The entire predicted amino acid sequences of human keratin 14 (HK14; [37]), Xenopus luevis XK81 (XK81; [24]), ON, [18] and human glial fibrillary acidic protein (GFAP; [32]) are compared to the predicted amino acid sequence of the GK50 cDNA. Amino acids identical to GK5O are in boldfuce. The conserved helical core regions are under and over lined. A conserved C-terminal domain characteristic of type I keratins is marked above by usterisks

Linker 1

Helix

Helix 2

Total

1B

Linker 2

H2

1A

68 70 72 68 65 40 43

36 27 33 27 43 8 8

68 67 64 60 52 28 30

60 60 47 47 20 21 6

60 54 56 50 43 33 37

30 33 30 32 34 5 16

54 52 49 46 40 25 23

Human keratin 15 (HK15; [29]), human keratin 14 (HK14; [37]), Xenopus lueuis keratins XK70 [66] and XK8l [24], human keratin 18 (HK18; [45]), ON3 [18] and human glial fibrillary acidic protein (GFAP; [32]) are compared to the GK5O protein

40

1 2 3 4

1.6kb

* Fig. 6. Blot-binding assay on goldfish optic nerve cytoskeletal proteins using in vitro transcribed and translated GK50 as a probe showing type I1 keratins. Positions of proteins O N , - 2 and proteins ON3- 4 are marked by a bracket

Fig. 5. Northern-blot analysis of GK50 mRNA. Total RNA was isolated from: lane I, goldfish optic nerve; lane 2, goldfish retina; lane 3, goldfish spinal cord; lane 4, goldfish brain; and probed with GK50 cDNA in a Northern-blot analysis

The GK50 protein is most similar in amino acid sequence to human keratins 14 and 15 (Table 1). These keratins are normally expressed in stratified epithelial tissue and have not been previously localized to nervous tissue [29, 371. We show that there are at least three distinct type I keratins in the nerve based on antibody reactivity (Fig. 1 B) and the ability to pair with a type I1 keratin in a blot-binding assay (Fig. 1C). There are also several type I1 keratins that bind to the type I keratin GK50 in a blot-binding assay (Fig. 6). The GK50 protein is expressed at a much lower level in the goldfish optic nerve than the ON, protein. Also, the ON3 protein is expressed in goldfish retina [19] while GK50 is not (Fig. 5). This, and their different patterns of expression in response to nerve injury (Fig. 7), implies that they are not components of the same keratin heterodimer since keratins are usually expressed in a cell-specific manner as specific keratin pairs [20]. Several studies have shown that GFAP is not in teleost optic nerve [9, 30,43, 511. Other studies have found that GFAP is in teleost optic nerve [6, 381 or that it is expressed only during regeneration [61]. We show a multiplicity of keratin expression in the goldfish optic nerve with a molecular weight between 44 kDa to 58 kDa. The complexity of keratin expression in the optic nerve is not limited to goldfish but is also found in other species of fish and amphibia [38, 39, 561. That GFAP is expressed in teleost optic nerve should be considered in light of the complexity of keratin expression in this tissue. The expression of keratins does not rule out the possibility of coexpression of GFAP. Keratins have been found to be coexpressed with GFAP in several other cell lines and tissues, such as in myoepithelial cells of salivary glands, pleomorphic adenomas [ 11, astrocytomas [8] and ependymomas [36]. We demonstrate in this and in previous studies [18, 191 that the predominant intermediate filament proteins in the goldfish optic nerve

are keratins. Other intermediate filament proteins, such as GFAP, must be in relative low abundance. Glial cells are closely associated with regenerating axons in the goldfish [67] and are thought to play a critical role in fiber channeling and pathfinding during axonal regeneration [68, 691. Many tissues expressing keratins in higher vertebrates provide a framework for migrating embryonic cells during development [65]. The glia of fish optic nerve form a unique superstructure and are different from glial cells found in other regions of the nervous system, including the adjacent optic tract [30, 34, 351. The astrocytes of the optic nerve in cichlid fish have been designated reticular astrocytes because of this specialized morphology [ 351. The reticular astrocytes have processes that fasciculate together and are linked by desmosomes [35]. This epithelium-like sheet composed of glial cells expressing keratins and connected by desmosomes has also been observed in amphibian optic nerves [56]. This is in contrast to optic nerve glial cell populations of higher vertebrates which express GFAP and lack desmosomes [49]. Keratins have not yet been identified in glial cells of higher vertebrates. An exception may be a sub-population of astrocytes in the rat optic nerve [56]. The unique structure and morphology of glial cells of lower vertebrate optic nerves and their expression of keratins may have evolutionary significance and reflect the neuroectodermal origins of the central nervous system [56]. Recent evidence suggests that the failure of axonal regeneration in the mammalian CNS, including the optic nerve, is not simply a property of the neurons but depends on the environment surrounding injured axons [4,5,16,27]. In fish optic nerve the environment through which the axons traverse is distinctive. The finding that keratins are the predominant glial filament proteins [ 18, 191 taken together with the elegant morphological analysis of Maggs and Scholes [35] support this view. The complexity of keratin expression is extensive (Fig. 1 and Fig. 6) in that at least three different type I keratins are observed in goldfish optic nerve. Furthermore, the localized pattern of keratin expression is different for the type I keratin GK50 and the type I1 keratin ON, (Fig. 7). The simplest explanation for this is that differ-

41

1 103nt-

2

3

1

B

2

3

D 373nt-326nt -110nt

Fig. 7. ON, and GKSO mRNA in the goldfish optic nerve during regeneration. A In situ hybridization with ON, cDNA 10 days post crush. The crush zone is marked by a brucket. (Magnification x 92). B RNase protection of ON, from total RNA: lane f, labelled probe; lane 2, protected fragment in uncrushed control; lane 3, protected fragment 10 days post-crush. Arrows designate the size of unprotected and protected fragments. C In situ hybridization

with GK50 cDNA 10 days post crush. The crush zone is marked by a bracket. (Magnification x 92). D RNase protection of GK50 from total RNA: lane f, labelled probe; lane 2, protected fragment in uncrushed control; lane 3, protected fragment 10 days postcrush. Arrows designate the size of the unprotected and protccted fragments

ent non-neuronal cell types within the optic nerve express different keratin pairs. Thus, in addition to distinct keratins in the optic nerve, comparison of G K 5 0 and ON, expression by the protection assay with the in situ hybridization analysis suggests a greater complexity of expression, with some keratins increasing only at the crush zone. Similar results have also been observed using antibodies to other goldfish optic nerve cytoskeletal proteins [31]. Besides astrocytes, the optic nerves of fish contain diverse cell types, including oligodendrocytes, microglia and large lipid-laden macrophages [l 1, 351. These cells may require expression of a specific keratin heterodimer to fulfill the physiological demands associated with lower vertebrate visual pathways. The finding that the G K 5 0 protein is found in optic nerve, brain and spinal chord but not in retina suggests that the cell type expressing this protein is highly specialized. These cells may be involved in ancillary processes associated with the growth of the optic axons through the crush zone. Studies involving the structural determination and cellular localization of these cell specific keratins (e.g. GK50 and ON,) will provide a molecular link between the mor-

phology of the glial types and their role both in nerve development and regeneration. Acknowledgements. We wish to thank Dr. Andrew Francis for critical review of this manuscript. This work was supported by a grant from the National Institutes of Health (EY 05212) to N.S.

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