The single lamprey neurofilament subunit (NF-180) lacks multiphosphorylation repeats and is expressed selectively in projection neurons

ELSEVIER

MOLECULAR BRAIN

RESEARCH Molecular Brain Research 29 (1995) 43-52

Research report

The single lamprey neurofilament subunit (NF-180) lacks multiphosphorylation repeats and is expressed selectively

in projection neurons

Alan J. Jacobs, John Kamholz, Michael E. Selzer *

Department of Neurology and Dacid Mahoney Institute for Neurological Sciences, Unicersity of Pennsykania Medical Center, Philadelphia, PA 19104-4283, USA

Accepted 27 September 1994

Abstract

The lamprey is considered the most primitive living vertebrate and its neurofilaments (NFs) are unique in being homopoly- mers of a single 180 kDa subunit (NF-180). Previous immunologic studies have suggested that the sidearm of NF-180 is highly phosphorylated selectively in the largest diameter axons. We report in this study the isolation and characterization of cDNA clones encoding the NF-180 lamprey protein. In situ hybridization with digoxigenin-labeled cRNA revealed NF-180 message exclusively in neurons with long axons, such as reticulospinal neurons and cranial motor neurons. The core of NF-180 was similar in structure to those of mammalian neurofilaments, but surprisingly, the carboxy sidearm lacked the multiphosphorylation repeats characteristic of higher vertebrate and invertebrate neurofilaments. Overall there was a paucity of potential phosphorylation sites in the NF-180 carboxy-terminus compared to NF-M and NF-H of mammals, fish and squid. This, along with the highly acidic nature of the NF-180 sidearm, makes it unlikely that phosphorylation of sidearm residues regulates interfilament spacing and axon diameter through global electrostatic repulsion of the carboxy-terminus away from the filament backbone. Further- more, the expression of a single neurofilament subunit in the lamprey that is most similar to the NF-M of higher vertebrates suggests that all three mammalian neurofilament subunits evolved from a single NF-M-like precursor.

Keywords: Intermediate filament; Phosphorylation; Evolution; Digoxigenin; cDNA

1. Introduction

Neurofilaments (NFs) are among the most prominent and highly conserved structures in the nervous system but their function remains unclear. NFs of most vertebrate and invertebrate species are heteropolymers of two or three subunit peptides, designated as’ low (NF-L), middle (NF-M) and high (NF-H) molecular weight based on size and immunologic characteristics [lo]. These subunits differ primarily in the length and composition of their carboxy terminus, which in NF-M and NF-H can be multiply phosphorylated [4]. A feature common to all NFs sequenced thus far is the presence of multiple phosphorylation repeats containing the amino acid triplet Lys-Ser-Pro. It has been proposed that phosphorylation of these repeats regu-

* Corresponding author. Fax: (1) (215) 573-2107.

0169-328X/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0169-328X(94)00227-4

lates axon diameter by electrostatic repulsion of sidearm domains away from the filament backbone [5,9,311. A striking piece of evidence in support of this hypothesis was the finding in the lamprey that mAbs specific for highly phosphorylated mammalian NFs selectively labeled the giant Miiller and Mauthner axons [31], whose diameters are many times the size of the largest mammalian axons [33].

Of the three mammalian NF subunits, only NF-L can self assemble in vitro into long stable intermediate filaments under physiologic conditions, while NF-M and NF-H require the presence of NF-L to form normal filaments [2,11,22]. In vivo, however, all three NF subunits are incapable of self assembly when expressed in cells lacking a preexisting IF network [6,20]. Assem- bly competence is restored when NF-L is coexpressed with either of the higher molecular weight subunits, but NF-M does not form filaments with NF-H. Both NF-L and NF-M appear early in neuronal develop-

44 A.J. Jacobs et al. /Molecular Brain Research 29 (1995) 43-52

A 10 30 50 70 90 110

GAATTCCATCACCACCACCACATCAACAGCCAGCCACAGCAGCAGCAGCATCACCAGCATCAGCAGACTCC~CAGCACCACCGTTCCTCCTCGCACACCACCACCACCACCCACCACCGCCAC

130 150 170 190 210 230 CATGAGCTACACAGAGTCGAGCGTGACGTGCTCCTACCGGCGCGCGTACGCCGACGCGTCCCGCGGTGGCAGC~GCGCCTGCTGAGCACTCCCTCCAGCGGGTTCCGCTCGCAGTCCTG MSYTESSVTCSY RRAYADA SRGGSKRLLSTPS SGFRSQSW

250 270 290 310 330 350 GTGCCGGGGCAGCAGCGGCGGCGGCTCCGGCTCCCTGGTGTCCCTGCAGAGGCGCCCCAACTACGCGAGCCTCTCGCACGGCTACAGCACCCCGGCCCTCGGCGGCGGACAGCCT C R G SSGGGSGSLVSLQRRPNYAS LSHGYSTPALNSAADSL

370 390 410 430 450 470 CGACTTCATGAGCCAGTCGAGCGTGTTCAGCGGGGACTTC~GCACTCGCGCGTC~CGAG~GCAGCTGCTGCAGGACCTC~CGACCGCTTCGCCGGCTACATCGAG~GGTGCACGA DFMSQSSVF SGDFKHSRVNEKQLLQDLNDRF A G Y I E K V H D

Head [o Core 0 Coil Ia 0 0 0 -

490 510 530 550 570 590 CCTGGAGCAGAAGAACAAGGAGCTCGAGACGGAGATCTCCGCCTACAGGCAG~GCAGTCGGGTCCCGCGCGGGGCGGCGGCGTGAGCGACGTCTACGAGCAGGAGATC~GGAGCTGCG LEQKNKELETEI SAYRQKQSGPARGGGV SDVYEQEI K E L R 0 +

' Coil Ia ’ o 0 0 Coil Ib o o . 4 610 630 650 670 690 710

AGACGTCATCGACGACATCACGGCGAGAAGACGACGACCGTGCAGATCGAGCAGGAGCACCTGGACGAGGAGATCCAGCGCCTGCGCGAGAAGACCGACGACGAGGTGCGGCTGCGCRACGA DVIDDINGE KTTVQIEQEHLD EEIQRLRE KTDDEVRLRNE

0 0 + 0 + “CoilIb” o + + +

730 750 770 790 810 830 GACGGAGGCGCTCATCAACGCGTTCAGGAAGAACGTGGACGACACGTCGCTGGTGCGCATGGAGATGGACAAGCGCACGCAGTCGCTGCTCGACGAGATCACCTTCCTCAAGAAGAACCA TEALINAFRKNVDDTSLVRMEMDKRTQSLLDE ITFLKKNH + cl cl 0 + + o Coil Ib + o 0 0

850 870 890 910 930 950 CGAGGAGGAGGTGGACGAGCTGCTCGCGCAGATCCAGTCGTCCACGGTGTCGGTGGAGAGGRAGGACTTCGCAGTGCCCGAGATCACGGCGGCGCTGCGCGAGATCCGCGGGCAGCTCGA EEEVDELLAQIQSSTVSVERKDFAVPEITAALREIRGQLE

0 Cl 0 0 0 Coil II + o ) 4 970 990 1010 1030 1050 1070

GGGGCAGTCGGCTCGCAACATCGAGACGGCCGAGGAGTGGTTC~GGGG~GTTCTCGCAGCTCACCGAGGCGGCCGAGCAG~C~CGACGCGATCCGCTCCGCC~GGAGGAGATCAC G Q SARNIETAEEWFKGKFSQLTEAAEQNNDAIRSAKEEIT

0 Cl + 0 cl o Coil II o + 0 Cl 0

1090 1110 1130 1150 1170 1190 GGAGCACCGACGCAAGCTGCAGATGCGCTGCACGGAGCTGGACGCCTTGGCCGGCACCAAGGAGTCGCTGGAGAGGCAGCTGAGCGAGATGGAGGAGAGACACCAGAGCGACGTGGGCAA EHRRKLQMRCTELDALAGTKESLERQLSEMEERHQSDVGN

0 0 + 0 0 + o Chill1 O o 0

1210 1230 1250 1270 1290 1310 CCTGCAGGATGCAGCCCAGCAGCTGGAGAACGAACTACGCAACACCAAGTGGGAAATGGCACGCCACCTGCGCGAGTACCAGGACCTGCTCAACGTCAAGATGGCGCTTGACATCGAGAT LQDAAQQLENELRNTKWEMARHLREYQDLLNVKMALDIEI 0 0 0 0 + 0 + Coil II o o + 0 0

1330 1350 1370 1390 1410 1430 CGCAGCATACdGGAAACTCTTGGATGGGGAGG~TCCGCTACAGCAGTGGCCCGCTGCCGACCCCGGCC~GCCGCCC~GGCCCCCTCGGC~GCCC~GGCTGCC~GGTCGAG~ AAYRKLLDGEEIRYSSGPLPTPAKPPKAPSAKPKAAKVEK

o Coil II o + Core ] Sidearm b

1450 1470 1490 1510 1530 1550 GAAAGTGGTGAGCAAGAAGCCAGAGATCAAGGTGGAGAGCGAACCAATCAGTGCTCAGCTTGACACCGACCTCGAAGACCTCGCACAGGAAGAGGTGATGGAGCAAAGGCAGCTCCGGT KVVSKKPEI KVESEPISAQLDTDLEDLAQEEVMEAKAAPV

1570 1590 1610 1630 1650 1670 GGTCAGTGCCGAGAAGGACGAAGAAGAAGAAGRGGRGGAGGAAGAGGACCGAGGCCGGAAGGAGGGAGAAGCTGAAGC VSAEKDEEEEEEEEEKEEEEAEAEEEEEEDRGRKEGEAEA

1690 1710 1730 1750 1770 1790 AGAGGAAGAAGCTGAAGAGGAGGTGGAGAAGGAGGAGGCAGAAGAAGCTGAAGTTGAGGAGGCTGAGGCGGAGGAGACAGAAGCTGAAGCAGCTGAAGAAGAGGAAGAAGCCGAGGGTGA EEEAEEEVEKEEAEEAEVEEAEAEETEAEAAEEEEEAEGE

1810 1830 1850 1870 1890 1910 GGAAGAGGCTGAGGCTGAAGGCGAGGAGGCGGAGGAGGCTG~G~GTTG~GAGG~GCCATTG~GGCAG~GCTGCAGAGGCT~GGCTGAGGTAGAGG~GAGG~GCAGAGGC EEAEAEGEEAEEAEEVEEEAI EKAEAAEAKAEVE EEEAEA

1930 1950 1970 1990 2010 2030 TGAAGAAGAGGAGGAGGAAGAAGCAGAAGAAGAAGAAGTTG~GCTG~CT~G~G~GTCGAGGCAGAGGCTG~GTTGAGG~GAGGGCG~GCAGCTGAGG~GAGGCAGAGGA EEEEEEEAEEE EVEAETKEEVEAEAEVEEEGEAAE E E A E E

A.J. Jacobs et al. /Molecular Brain Research 29 (1995) 43-52 45

ment, at about the time when postmitotic neurons begin to grow processes [5]. It has been speculated that the three mammalian NF subunits evolved by duplica- tion of an ancestral intermediate filament (IF) similar in structure to the types I-III IF proteins, followed by

the lengthening and divergence of the carboxy-terminus [19,391. However, NFs of the most primitive living vertebrate, the lamprey, are composed of a single higher MW subunit (NF-180) [19,311 and thus far, no chordate species has been identified in which NFs are composed

2050 2070 2090 2110 2130 2150 AGAAGAGGCAGAGGAAGAAGAAGTAACTTCAAAAAA GGCTAAAACACAAGAAGCTGAAGTTGAGGAAGAAGAGGCTGAAGCAGCAGAGGCTGAAGCTGAGGAGGAAGCTGAAGAAGAAGC EEAEEEEVTSKKAKTQEAEVEEEEAEAAEAEAEEEAEEEA

2170 2190 2210 2230 2250 2270 TGGAGAGGAGGACGTTGAAGCTGAATCCAAAGAGGAGGAAGCGGACGCTGAAGAAGACGAAGCAGAAGAAGAGGAGGTTAAAGAAGAAGAGGTCACCACC~GTC GEEDVEAESKEEEEEDSKEADAEEDEAEEEEVKEEEVTKS

2290 2310 2330 2350 2370 2390 AGATGCTGAAGAAGCTGAGGCTGAAGCAGAAG~G~GAGGCTGCC~GTCAGAGGAGG~GCGGCTG~G~GCC~GATG~GCTGAGGAGG~G~GCAGAGGAGGAGGCTGTTG~GA DAEEAEAEAEEEAAKSEEEAAEEAKDEAEEEEAEEEAVEE

2410 2430 2450 2470 2490 2510 AACTGAAGCAGCAACTGAAGAAGCTGAAGCAAAGGAGGCCTCAGACGATGAGAAACCCGAGGAAGAGGTGAAAGAGTCAGAGGCCCCTGTTGCACCTGAAGCTAAGAAAGCCCCAGAGCC TEAATEEAEAKEASDDEKPEEEVKESEAPVAPEAKKAPEP

2530 2550 2570 2590 2610 2630 AAAAGCAGCTCCCAAAAAGAAGGCACCCGCAGGTGGAGTCACCCACGTCTGAGCCAGAGGATGRACCAGCAGAAGTTGTTGAGAAGGCTGAAGCTCCCAAACCCAA KAAPKKKAPAKVESPTSEPEDEPKAEVVEKKGKAEAPKPK

2650 2670 2690 2710 2730 2750 GGCCAAACCTGCAGCTGCCAG~GGAAGCCAAGCCGGTGGAAAAGGAAGAGGAACCGGAGGAGG~TCCCCCACTGAGGAAGAACCGAAGAAACCTGCTGCCGCTAAGCCTGCCAAGGCCCC AKPAAAKKEAKPVEKEEEPEESPTEEEPKKPAAAKPAKAP

2770 2790 2810 2830 2850 21370 TGCCAAGCCCAAGCCCGCTCCCAAAGCAGAAGCAGAGGAGACCAGAACCAGCGAAACCCGCCCAGGCGAAGCCTGCGCCAGCTGCCGAGGAGGAGGAGGATGAGAAGGAGGATGATGA AKPKPAPKAEAEEKPEPAKPAQAKPAPAAEEEEDEKEDDE

2890 2910 2930 2950 2970 2990 GGAAGAGGAGGAGGAGGTGGAGGAAGTGAAACCTGAGGACGCCAAACCCGTCAAGTCAAAGCCCGCCCCGGCCAAAGAGGAGGAAGATGAACCCC~CCCGCC~CAGCCACCC~GCC EEEEEVEEVKPEDAKPVKSKPAPAKEEEDEPKPAKQPPKP

3010 3030 3050 3070 3090 3110 CAAACGGAAGCCCGCTCGTCCTAAAGAGGAGCCAGAAGACAGCAGAGCCGGCGRAGGAAAAACACTCACCGGTCGAGGAGCGGAAGCCCATCAAAGAGATCGCCAAACCTGCAAAAGC KRKPARPKEEPEDKAEPAKEKHSPVEERKPI KEIAKPAKA

3130 3150 3170 3190 3210 3230 GGCACCGGCAAAGGCTGACAGAGCCAGAAGCGGCCGAGGCAGAGCACGCAGCAGGAGGA APAKADKEPEAAEPKKIEVKVKKVTKKVVEEIEQSTQQEE

f-- 3250 3270 3290 3310 3330 3350

GTCGTACGAGGAGATCATCGAGGAGACGATCGTCTCCACAAAGGGTGGCGGCCCCGAGCAAGGAGCCCAAGAAAGCCGTCAAGGTGAAGGAGGAGGCGCCCAAGGCTGTGGC SYEEII EETIVSTKKVEKVAAPSKEPKKAVKVKEAPKAVA

Repeat 1 --------_-------_* 3370 3390 3410 3430 3450 3470

CCAGGTGGAGGAGTACGAGGAGATCATCGAGGAGACGGTGGTGTCCACCACCAAGAAGCACGAGAAGGGCAGGCGCCTGTCGATTCCAAGTGAGCCCTGGGAGAGGATTCGATGCCATT QVEEYEEIIEETVVSTTKKHEKGKAPVDSK'

Repeat 2 * ______________ ----) 3490 3510 3530 3550 3570 3590

CGCTCAGTTACTGAGCATTGTCCAAAAGTGCAATACCGAGCCATGACGACAACAACACAGAACCCAGACCATACCCTACGATAAGAGTCAAACACTTGCACTATTAAGTTTACTTTTTRA

3610 3630 3650 3670 3690 3110 TCGATTATGCTGCATTGATGTTTCTGCTCGTAATGCTTGCAGAGACCACCGTTACCTTATGTGTTGTGCATGT~TACATT~GTC~TT~TATT~G~CAG~TGTT~

3730 3750 AAAAAAAGAAAGGGGCCCTTGATGATCCACCAGATCTCGTGGT

Fig. 1. A: nucleotide and predicted amino acid sequence of the Lamprey neurofilament protein NF-180 derived from the assembly of cDNA clones LIFS, LIF13 and LIF22. Translation was begun at the first in-frame methionine of the longest open reading frame. Nucleotide numbers are indicated above the DNA sequence. Junctions between head, core and sidearm domains are indicated with brackets. Open circles mark hydrophobic residues at the first and fourth amino acid of putative heptad repeats, while crosses mark charged residues. Three predicted heliu-forming regions (coils Ia, Ib and II) are indicated by arrowed lines. A 27 amino acid repeat at the carboxy terminus is indicated by dashed arrows and the labels Repeat I and Repeat 2. A potential polyadenylation signal sequence is double underlined in the 3’ untranslated region, B: schematic drawing of NF-180 indicating relative sizes and overlap of the cDNA sequences from clones LIFS, LIF13 and LIF22. Predicted 5’ and 3’ untranslated regions of the cDNAs are indicated by dashed lines. Glutamate-rich and lysine-proline rich domains are indicated by E and KP, respectively. The polyadenylation sequence in clone LIF13 is indicated by AAAAA. Numbers correspond to nucleotides in DNA sequence.


B

Head lOLD

Core a6.s LD

Sidearm 76 LD

Proline Gl”tPmPte Kp E KP Rich Rifh Rich Rich Rich

loby 3688

I Lnw

I ,948 17M

Fig. 1. (continued).

solely of NF-L [19,26,38]. Thus, it remains unclear whether all three mammalian NF subunits evolved from an NF-L-like precursor similar in structure to non-neuronal intermediate filaments such as desmin and vimentin, or whether they arose from a higher MW form already possessing a long carboxy-terminus sidearm and a self assembling core. Based on fossil records, the lamprey has retained features of the earliest vertebrates and thus may occupy a pivotal position close to the root of the vertebrate phylogenetic lineage. We report here the isolation and characterization of overlapping cDNA clones that encode the lamprey NF-180 protein.

2. Materials and methods

2. I. Screening of cDNA library and nucleotide sequence analysis

A random hexamer primed lambda-gtll cDNA library constructed from polyadenylated RNA isolated from the brains of larval wild-type Petromyzon marinus lampreys was screened with a a*P- labeled cDNA fragment of the human NF-L (HUNFL.5.1) [17]. Duplicate lifts of one million plaques yielded 37 positive phage clones, two of which, LIFS and LIF22, were found by sequence analysis to encode partially overlapping regions of the same protein. Sequencing was performed by the dideoxynucleoside method [36] on nested deletions [35] with synthetic oligonucleotide primers using a Sequenase 2.0 kit (U.S. Biochemicals, Cleveland, OH) as described by the manufacturer. Sequences were determined for both cDNA strands. Nucleotide and amino acid sequences were analyzed using the DNA Strider (Departement de Biologie, Commissariat a I’En- ergie Atomique, France), LFAST and FASTA [29] computer pro- grams. Nucleotide sequences of other intermediate filament and lamin proteins were obtained for comparison from the GenBank data base (Los Alamos, NM).

2.2. Northern blot analysis

For analysis of mRNA expression, total RNA was prepared from larval wild-type P. marks CNS, liver and muscle by the acid guanidinium thiocyanate-phenol-chloroform extraction method [7]. Aliquots of 5 pg were separated on formaldehyde agarose gels [35], capillary eluted to nitrocellulose membrane, hybridized to probes labeled with ‘*P by random primed synthesis, and washed at high

stringency (0.2 X SSC, 65°C). The blots were then exposed to autora- diography film for I week.

2.3. In situ hybridization with digoxigenin-labeled riboprobes

Digoxigenin-labeled riboprobes constructed from isolated clones were hybridized to sectioned and whole-mounted lamprey brainstem, essentially as described 1421. Briefly, for whole-mount preparations brains were removed, stripped of overlying meninx primativa and choroid plexus, pinned flat on Sylgard strips, fixed in 2% paraformaldehyde, washed in phosphate-buffered saline (PBS) and stored in 70% ETOH at 4°C. Tissue for sectioning was fixed in 2% paraformaldehyde, washed in PBS, dehydrated in serial ethanols, cleared in toluene and infiltrated with Paraplast. Digoxigenin-labeled cRNA riboprobes were constructed from subclones of NF-180 cDNA encompassing the long carboxy-terminal sidearm (i.e. LIF13). In vitro transcription was performed with an RNA transcription kit (Strata- gene, La Jolla, CA) as recommended by the manufacturer except for the inclusion of digoxigenin-11-UTP (Boehringer Mannheim, Indi- anapolis, IN) in a 35 : 6.5 ratio with unlabeled UTP. Transcripts were fragmented into approximately 100 nucleotide polymers by incuba- tion with sodium carbonate (0.1 M Na,CO,, 65°C 85 min) and ethanol precipitated. Both whole-mounted and sectioned tissue was hybridized to digoxigenin-labeled riboprobes as described [42].

2.4. Immunologic analysis of fusion proteins

Fusion proteins were constructed by insertion of portions of isolated cDNA into the cloning site of bacteriophage lambda gt-1 1. These recombinant proteins were screened for immunoreactivity with mAbs specific for mammalian NFs and ones specific for NF-180. YlO90 E. Coli were infected with recombinant lambda gt-I I, plaques grown overnight at 37°C and then overlaid with Duralose-UV membranes (Stratagene, La Jolla, CA) impregnated with isopropylthio+- o-galactoside (10 mM). These membranes were then probed with mAbs by the peroxidase-antiperoxidase (PAP) method [21].

3. Results

Screening of a P. marinus brain cDNA library at low stringency with a human NF-L cDNA probe [17] led to the identification and isolation of several phage clones, that strongly hybridized to human NF-L at higher stringency. One of these clones, LIF22 (2.7 kbp insert), was sequenced and found to contain a putative in-frame start ATG codon, a partially completed open reading frame that was very similar to human NF-M, and a 115 bp 5’ untranslated region. Two additional clones (LIFS and LIF13) representing the mRNA encoded by LIF22 were obtained by rescreening the library with a probe to the 3’ terminus of LIF22. The sequence of clone LIFS (2.7 kbp) overlapped with the 3’ end of LIF22 and contained a single 2.4 kbp open reading frame terminating with the putative TGA stop codon. Clone LIF13 (2.1 kbp) extended further 3’ of LIF5 and contained a potential polyadenylation signal ATTAAA (nucleotides 3679-3684) followed by a poly(A) tract (Fig. 1).

Assembly of these clones predicted a 124 kDa peptide containing features characteristic of NF proteins


Table 1 Amino acid identities (%I between NF-180 and various intermediate filament proteins Amino acid sequences of the head, core and sidearm domains of each IF protein were aligned to NF-180 and percentage identities calculated using the LFAST computer program 1291. Nucleotide sequences intermediate filament and lamin proteins were obtained from the GenBank data base (Los Alamos, NM).

Intermediate filament protein Head Core Sidearm

Type III Human Desmin Mouse GFAP Human Peripherin Chicken Vimentin Rat cY-internexin Xenopus Neuron al IF (XNIF) Torpedo Californica Type III Goldfish ON3

Type IV NF-L

Human Mouse Pig Quail Xenopus Squid (NF-60)

NF-M Human Mouse Chicken Goldfish Pacific Ray

NF-H Human Mouse Squid (NF-220)

Type V Human Lamin A Chicken Lamin B2 Xenopus Lamin A

Type VI Rat Nestin

15 19 21 30 28 17

6 12

14 27 28 31 10 23

38 43 44 32 17

13 12 23

12 13 10

7

50 2 46 5 49 9 50 2 50 3 54 2 47 3 37 5

52 6 52 6 52 6 53 7 50 7 23 6

63 19 63 16 61 16 57 16 59 13

52 22 52 10 23 13

24 7 25 2 20 6

14 17

(i.e. central alpha-helical rod domain, glutamic acid-rich tail domain). It showed a high degree of homology to middle molecular weight mammalian NF subunits. When the predicted amino acid sequence of this composite cDNA was aligned with human NF-M, the central core domain was 63% identical (Table 1). This degree of identity is greater than that seen between Torpedo fish [23] and human NF-M [27] and between any of the squid NF subunits [43,441 and human NF-M. The composite lamprey cDNA contained 48% charged residues, 32% acidic (Glu, Asp) and 16% basic (Lys, Arg and His), resulting in a predicted p1 of 4.18.

Comparable to other intermediate filament protein sequences [45], the 311 residue central rod domain of this putative lamprey NF cDNA contained a quartet of heptad repeats (lA, lB, 2A, 2B) in which 74% of the first and fourth amino acids were either non-polar or uncharged. However, within the segment linking he-

lices 1A and 1B (Ll) there were three glycine residues not found in any of the mammalian, amphibian or fish NF sequences investigated thus far. The carboxyl end of the rod domain was punctuated by the sequence YRKLLDGEE which, except for the conservative substitution of aspartate for glutamate, is highly conserved among intermediate filament proteins. A computer search of the latest update at NCBI using the BLAST [l] network service failed to find any intermediate filament protein containing this substitution. That this substitution is not found in intermediate filament sequences evolutionarily preceding or following NF-180 suggests its acquisition occurred after the divergence of agnatha from ancestral chordates.

The long carboxy sidearm of this putative lamprey NF cDNA, while less similar than other regions of the protein, shared the glutamic-acid rich nature of human NF-M but was devoid of its characteristic multiphosphorylation repeats. It was comparable in length to mammalian NF-H although the amino acid sequence was only 30% identical. The sidearm of the lamprey cDNA was comprised of a short proline-rich segment adjacent to the core, followed by two glutamic acid-rich regions interdigitated with a pair of lysine-proline-rich segments, and capped by a short neutral region at the carboxy terminus (Fig. 1A). Glutamic acid residues comprised 59% of the first acidic domain, mostly oc- curring in clusters of three to seven consecutive residues. Chou and Fasman analysis predicted an alpha-helical structure for this region. Following the first acidic domain was a region heavily populated by lysine-proline pairs. Conspicuously absent were the characteristic lysine-serine-proline (KSP) repeats so prominent in the sidearms of the higher molecular weight NFs of other species. Overall, the central region of the sidearm contained only 12 potential serine-phosphorylation sites while clusters of six serines were located at the extreme carboxy-terminus and adjacent to the core. Also located at the extreme carboxy- terminus were two 27-amino acid repeats (repeat 1 and 2; Fig. lA), having 74% identity with each other. They were comparable in location, but not sequence, to similar-sized repeats in mammalian NF-M [37]. Thus, the amino acid sequence of this composite cDNA suggested that it represents the mRNA of the lamprey 180 kDa neuronal intermediate filament protein.

The mRNA represented by overlapping clones LIFS and LIF22 was conclusively identified as encoding the previously characterized lamprey NF protein [19,311 by immunologic characterization of clone LIF13, which contained a partial cDNA of the long carboxy terminus sidearm. Infection of E. coli Y1090 with a h-gtll bacteriophage containing in-frame LIF13 cDNA, resulted in the expression of a fusion protein that immunoreacted with several anti-rat NF mAbs (courtesy of V.M.Y. Lee) and with mAbs that selectively recog-


Liver Muscle CNS

28s >

18s >

Fig. 2. Immunoreactivity of fusion protein encoded by LlF13. A: dot blot of fusion protein expressed by E. coli infected with bacteriophage A-gtl I containing in-frame cDNA of NF-180 sidearm (LIF13) or out-of-frame cDNA of NF-180 head and core (LIF22). A fusion protein encoded by LIF13 immunoreacted with mAbs that recognize sidearm epitopes of NF-180 (RM0270 and LCM39) but not with core specific mAbs (RM014 and LCM3). LIF22 cDNA inserted into bacteriophage in an incorrect reading frame resulted in a fusion protein that did not immunoreact with these mABs. B: immunoblot of lamprey CNS cytoskeletal proteins showing immunoreactivity of these mABs with NF-180.

LIF13 LIF22

RM014

RM0270

nize NF-180 on Western blots (Fig. 2). These antibodies also selectively label neuronal elements on tissue sections. That LIF13 encoded the sidearm portion of NF-180 was further demonstrated by the failure of core domain-specific anti-NF mAbs to detect the fusion protein, while mAbs that recognize non-phosphorylated sidearm epitopes immunoreacted with the protein. These immunoreactivities are in accordance with the previously described immunological characteristics of NF-180 [31]. Thus, based on the NF characteristics of the translated amino acid sequence and immunore- active properties of a fusion protein expressing the sidearm domain, we concluded that the cDNAs described here represent the mRNA encoding the lamprey NF-180 peptide.

Intermediate filament proteins are prominent components of the cytoskeleton and karyoskeleton that generally show tissue-type and developmental-specific patterns of expression (see ref. [39] for review). The tissue-specific expression of NF-180 in the larval lamprey was initially determined by Northern blotting. Complementary DNA probes specific for the carboxy- terminus sidearm of NF-180 hybridized to a prominent 3.7 kb band only in the CNS, but not to non-neuronal tissue (Fig. 3). Probes constructed from a cDNA encoding only the head and core domain of NF-180 (i.e. LIF221 also hybridized at high stringency to this CNS band. Thus, NF-180 appeared to be present in a single isoform with expression restricted to the nervous system.

B NFlSO)

LCM3

LCM39

Fig. 3. Northern blot analysis of tissue-specific distribution of NF-180 mRNA. Ahquots of 5 mg of total RNA from pooled CNS, liver and muscle of 20 wild-type P. marinus larvae were separated on 1% denaturing agarose gels, capillary blotted to nitrocellulose membrane and hybridized with a cDNA probe specific to the carboxy-terminus sidearm of NF-180 as described in Materials and methods. A conspicuous 3.7 kb band was detected only in the CNS. Positions of rat 28s and 18s ribosomal RNA are as indicated. Blot was exposed to autoradiographic film for 7 days.

A.J. Jacobs et al. /Molecular Brain Research 29 (I 995) 43-52

-

Fig. 4. Distribution of NF-180 mRNA in larval lamprey brainstem wholemounts and spinal cord sections determined by in situ hybridization with digoxigenin-labeled cRNA probes. A: in the brainstem NF-180 message was found exclusively in neurons with long projection axons, such as rhombencephalic identified neurons B,, B,, B,, I, and the Mauthner (Mth) neuron. Abundant labeling also occurred in neurons of the trigeminal motor (V,), facial (VII) and glossopharyngeal (IX) nuclei. B: in the spinal cord expression was detected in neurons of virtually all 8 wrn serial sections. Dorsal cells (DC) and motoneurons (MN) were uniformly labeled as were edge cells and several unidentified neurons. Expression was absent from ependymal cells surrounding the central canal (asterisk) and neuroglia. Labels for identified neurons in B are immediately adjacent to cells. Bars = 100 Km.

Cellular expression of NF-180 mRNA was analyzed by in situ hybridization with digoxigenin-labeled cRNA probes constructed from the carboxy-terminus of NF- 180. Sidearm rather than core sequences were used because this portion of the protein distinguishes NFs from all other intermediate filament proteins. In situ hybridization to whole-mounted larval lamprey brains indicated that NF-180 mRNA was abundant in all of the identifiable giant Miiller and Mauthner cells, in the majority of spinal-projecting neurons, and within neurons of the cranial motor nuclei (Fig. 4). NF-180 expression was detected uniformly throughout the cyto- plasm and in some neurons extended into the axon hillock and primary dendrites. Even under the conditions of intense of labeling illustrated in Fig. 4, expression was not detected in the telencephalon, olfactory lobes or optic tectum. Labeling was similarly absent from ependymal cells, areas rich in glial cell bodies such as fiber tracts of the posterior commissure and habenulo-peduncular tract, and the small interneurons located throughout the CNS. Under conditions of less intense labeling, the degree of staining remained simi-

lar between large and small reticulospinal neurons. No cells were labeled by NF-180 sense RNA probes.

4. Discussion

Unlike the triplet NF subunits found in mammalian neurons, lamprey neurons express a single 180 kDa subunit [193. Immunochemical analysis with mAbs specific for each of the three mammalian NF subunits revealed only one NF subunit in the lamprey which has core and sidearm epitopes characteristic of each mammalian subunit [311. Several observations confirm that the cDNA sequence described here encodes the NF-180 protein. (1) Deduced amino acid sequence of these clones revealed an alpha-helical rod domain that was most similar to the mammalian NF-M, and also shared significant homology with NF-L and NF-H. (2) The carboxyl end of the rod domain contained the highly conserved amino acid sequence YRKLLEGEE (except for one conservative substitution, see Results). This linear sequence includes the epitope for the anti-IFA


antibody [24], a monoclonal antibody specific for most vertebrate and invertebrate intermediate filament proteins [32], which immunoreacts with the NF-180 peptide [31]. (3) Although the molecular weight calculated from the cDNA sequence (124 kDa) was somewhat smaller than that suggested by SDS-PAGE (180 kDa1, electrophoresis is known to overestimate the molecular masses of NF peptides, presumably owing to their unusually acidic carboxy terminal domain [18]. (4) Im- munochemical analysis of a fusion protein containing the predicted long carboxy sidearm revealed a protein which immunoreacted selectively with mAbs specific for the sidearm of NF-180 protein. (5) Northern analysis and in situ hybridization demonstrated mRNA expression restricted to central nervous system neurons, as is NF-180 immunoreactivity. Based on these findings we conclude that the assembled cDNA sequence pre- sented here represents the mRNA encoding the lamprey NF-180 peptide.

4.1. Selectil:e expression of NF-180 mRNA in projection neurons

NF-180 message was expressed primarily in brainstem neurons whose axons projected into the spinal cord or out nerve roots, i.e. reticulospinal neurons and cranial motor nuclei. These neurons, however, represent less than 10% of the total neurons in the lamprey brainstem [34]. Despite the wide range of axon diameters in the reticulospinal neurons, labeling for NF-180 message was quite homogeneous. Under various hybridization stringencies, the intensity of labeling for NF-180 mRNA in the large Miiller and Mauthner reticulospinal neurons, whose axons can attain diameters of 50-100 pm, appeared similar to that in the smaller reticulospinal and cranial motor neurons, some of whose axons have diameters less than 1 pm. Thus, while the degree of NF-180 expression seemed to be independent of axon caliber in these cells, its message was found only in neurons whose axons extend sub- stantial distances within the CNS or into the PNS.

4.2. NF-180 sidearm is phosphorylated but lacks the multiphosphotylation repeats characteristic of higher MW NFs

The cDNA sequence of NF-180 demonstrated a protein with the defining characteristics of a neurofilament, including an alpha-helical central rod-like domain, a serine rich amino-terminal domain (23%) and a long highly acidic carboxy-sidearm with several serines that are potential sites of phosphorylation. The glutamic acid-rich portion of the NF-180 sidearm was considerably longer than those of described mammalian, avian, amphibian, fish and cephalopod NFs. Absent from NF-180 was the tandem-repeat motif containing multiple potential serine-phosphorylation sites present in other higher molecular weight NFs. Human

NF-M, for example, carries six nearly perfect repeats of a 13 amino acid sequence containing two sets of Lys-Ser-Pro [27]. Analagous multiphosphorylation repeats are also present in Torpedo califomica NF-M, except that this molecule contains 13 tandem repeats of six amino acids, each containing Lys-Ser-Lys and no prolines [23]. Goldfish NF-M is similar to NF-180 in that it does not have tandem multiphosphorylation repeats, athough its sidearm does contain three copies of a quintet and two copies of a quartet, each with lysine, serine and proline [12]. Lamprey neurofilament is thus far the only higher MW NF shown by amino acid sequence to be entirely devoid of tandem phosphorylation repeats. Squid NFs, which are postulated to have evolved from a separate lamin-like precursor, also possess repeated copies of a Lys-Ser-Pro motif 1441. The absence of multiphosphorylation repeats in the lamprey suggests either that such repeats evolved independently in invertebrates and vertebrates or that the lamprey lost this feature through divergent evolution. The recent demonstration of an intermediate form of NF sidearm in the goldfish containing scat- tered phosphorylation repeats of variable length and sequence, coupled with the presence of non-proline- containing multiphosphorylation repeats in Torpedo, suggests that higher vertebrates acquired multiphosphorylation sites through additions to a precursor protein similar to that of NF-180.

Immunohistochemical analysis of NF-180 had suggested the existence of a multiphosphorylation repeat region based on the cross-reactivity of phosphorylation- dependent NF specific mAbs, reactivity which was abolished by alkaline phosphatase treatment [31]. The small number of phosphorylation sites found in NF-180, correlates with the previously reported failure of phosphatase treatment to significantly alter the elec- trophoretic migration of NF-180 [31]. A mAb that recognizes a non-phosphorylated linear sequence of four amino acids (i.e. KSPV/A) within the multiphosphorylation repeat domain of mammalian NF-M and NF-H also immunoreacted with NF-180 [31]. However, this linear sequence was absent from NF-180 cDNA as were other sequences suggestive of tandem phosphorylation repeats. Presumably these antibodies react with lysine-serine or serine-proline doublets that are brought into proximity with other amino acids of the epitope through tertiary folding.

During development and regeneration, maturational increases in the diameters of mammalian peripheral nerve axons is accompanied by increases in NF phosphorylation [3,8,14,21,28,30,401. In dendrites, NFs are less phosphorylated, have shorter cross bridges and are more closely packed [15]. These observations led to the hypothesis that NF sidearm phosphorylation regulates axon diameter by controlling interfilament spacing through a mechanism involving electrostatic repulsion


of phosphorylated sidearm domains from each other and from the filament core [5,31]. However, in vitro removal of up to 90% of NF-H phosphates by alkaline phosphatase did not reduce the extended sidearm configuration of NFs or their ability to form cross-bridges [16]. For NF spacing to be modulated by electrostatic repulsion, the charge added by linkage of phosphate residues should be significantly greater than the native (unphosphorylated) charge of the sidearm, otherwise addition of phosphates would not increase net charge sufficiently to change repulsive interactions. In mammalian and invertebrate NFs, phosphorylation of residues in multiphosphorylation repeat domains produces a dramatic increase in total sidearm charge. The ratio of phosphorylatable sidearm residues (i.e. serine) to acidic and basic residues in human NF-M, mouse NF-H, and squid NF-H is 1.0:3.7:2.4, 1.0:2.0: 1.8 and 1 .O : 0.9 : 0.6 respectively. Net unphosphorylated charge is therefore close to neutral and the addition of phosphate moieties would confer a significant negative charge. However in the lamprey, the native NF sidearm is highly acidic (1.0 : 11.1 : 4.2 ratio serine to acidic to basic residues). Phosphorylation of the few NF-180 serines would have little effect on overall charge char- acter. It is therefore unlikely that phosphorylation of NF-180 alters sidearm conformation primarily by global electrostatic repulsion. Yet in the lamprey, immunohis- tochemistry has demonstrated axon specific phosphorylation of NF-180, and expression of distinct phosphorylated isoforms exclusively in the largest diameter axons. It is possible that phosphorylation of NF-180 produces sidearm extension by localized ionic or steric effects. Tail domains of the higher MW NF subunits are segre- gated into highly acidic and basic regions. Ionic interactions between these regions may cause the sidearm to take on a folded accordion-like configuration which is disrupted by the coupling of phosphates to serine residues within the basic (lysine-rich) domains.

4.3. Evolution of all three mammalian NF subunits from an NF-M-like precursor

The lamprey is considered the most primitive living vertebrate and as such occupies a pivotal position near the root of the vertebrate phylogenetic lineage (re- viewed in ref. [13]). Recent molecular analysis of lamprey and hagfish 18s ribosomal RNA is consistent with this view [41]. Neurofilaments of all mammals studied thus far are heteropolymers of a triplet of low, middle and high MW subunit proteins. The same is true for most birds and amphibians (except parakeet and newt, which appear to lack NF-H). Lower vertebrates are more heterogeneous in the size and number of subunits that are assembled into neurofilaments. Reptiles, for example, have only two subunits of low and middle MW, as do sharks, rays and some teleosts 119,261. Thus,

the minimum complement of higher vertebrate NFs includes a low MW subunit and at least one higher MW isoform. No chordate species has yet been found with just NF-L. However, NFs of the lamprey and three protochordates (Ciona intestinalis, Branchios- toma lanceolatum and tunicates) appear to be ho- mopolymers of a single middle MW subunit [19,25]. The absence of a low molecular weight NF subunit in sub-vertebrate chordates and in the most primitive vertebrate, the lamprey, suggests that all three vertebrate NF subunits evolved from a middle MW precursor rather than from a small cytoplasmic intermediate filament protein similar to desmin, vimentin or NF-L [19,39]. The finding of epitopes unique to NF-L within the NF-180 peptide [31], supports the proposal of NF- 180 as the precursor molecule to NF-L. Multiple ge- netic duplications and selective loss or elongation of the long carboxy-terminus is one mechanism for deriva- tion of lower and higher molecular weight subunits from an intermediate sized precursor. The presence of a long carboxy-terminus in the earliest vertebrate NFs and its persistence in higher vertebrates suggests that the sidearm of NF serves an essential and unique neuronal function.

Acknowledgements

We thank Drs. Rachael Neve, Garth Hall and Ken- neth Kosik for supplying the lamprey CNS cDNA library. This research was supported by NIH Grant NS14837. A.J.J. was supported as a Medical Student Research Fellow of the American Heart Association and is currently an NIH MSTP Trainee, Grant 5-T32- GM07170.

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The single lamprey neurofilament subunit (NF-180) lacks multiphosphorylation repeats and is expressed selectively in projection neurons