Domain structure of neurofilament subunits as revealed by monoclonal antibodies

Monoclonal antibodies have been prepared against purified neurofilament (NF) subunits (NF68, NF150, and NF200). From 25 fusions, several hundred strongly positive antibodies have been obtained. Among them are antibodies against the specific subunits as well as antibodies recognizing common antigenic determinants. These have all been characterized according to the following properties: ELISA (enzyme-linked immunosorbant assay) testing against each subunit, immunoblots against enriched neurofilament preparation, immunoblots of cyanogen bromide or chymotrypsin-treated neurofilaments, immunofluorescence with PC12 cells, and immunohistochemistry of cerebellum. Whereas the antibodies against the NF68 and NF150 appear to react with single cyanogen bromide fragments, the antibodies against the NF200 react with multiple cyanogen bromide fragments. These data are consistent with the hypothesis that the NF200 is partially composed of several repeated structural determinants. Furthermore, all of the antibodies that react with the NF200 recognize the solubilized "sidearm" domain from limited chymotryptic digestions. The locations of the common and variable domains of the three subunits are discussed in light of these results.     

Defective axonal transport of neurofilament proteins in neurons overexpressing peripherin

Peripherin is a type III neuronal intermediate filament detected in motor neuron inclusions of amyotrophic lateral sclerosis (ALS) patients. We previously reported that overexpression of peripherin provokes late-onset motor neuron dysfunction in transgenic mice. Here, we show that peripherin overexpression slows down axonal transport of neurofilament (NF) proteins, and that the transport defect precedes by several months the appearance of axonal spheroids in adult mice. Defective NF transport by peripherin up-regulation was further confirmed with dorsal root ganglia (DRG) neurons cultured from peripherin transgenic embryos. Immunofluorescence microscopy and western blotting revealed that excess peripherin provokes reduction in levels of hyperphosphorylated NF-H species in DRG neurites. Similarly the transport of a green fluorescent protein (GFP)-tagged NF-M, delivered by means of a lentiviral construct, was impaired in DRG neurites overexpressing peripherin. These results demonstrate that peripherin overexpression can cause defective transport of type IV NF proteins, a phenomenon that may account for the progressive formation of ALS-like spheroids in axons.     

Chromosomal localization of the mouse gene coding for the 68 kDa neurofilament subunit

The chromosomal localization of the mouse gene coding for the 68 kDa intermediate filament subunit of neurones (NF-L) was determined by in situ hybridization using specific 3H-labelled DNA probes. There is only one copy of the NF-L gene. The gene encoding NF-L is located on chromosome 14 region (D1-E1).     

Complete separation of the triplet components of neurofilament by DE-52 column chromatography depends upon urea concentration

In the separation of the triplet components of neurofilament (P 200, P 160, and P 68) by DE-52 column chromatography in the presence of urea, it was revealed that the efficiency of separation depended upon urea concentration. When chromatography was performed in the presence of 8 m urea and a linearly increasing sodium phosphate concentration from 10 to 400 mm at pH 6.8, P 160 and P 68 were eluted in the same peak, although P 200 was eluted faster. P 160 and P 68 were partially separated with 6 m urea, and completely separated with 4 m urea. But, under these conditions, P 200 was eluted at the same position as contaminated glial fibrillary acidic protein (GFA). From these results, two methods were recommended for the complete separation of the triplet components of neurofilament and GFA by DE-52 column chromatography. In one method, chromatography was performed in the presence of 8 m urea at first, and then P 160 and P 68 were separated in the presence of 4 m urea. In the other method, chromatography was performed with a linearly decreasing urea concentration from 8 to 0 m.     

Type II keratin cDNAs from the rainbow trout: implications for keratin evolution

From a teleost fish, the rainbow trout Oncorhynchus mykiss, we have cloned and sequenced cDNAs encoding five different type II keratins. The corresponding protein spots, as separated by 2D-PAGE of trout cytoskeletal preparations, have been identified by peptide mass mapping using MALDI mass spectrometry. Three of the sequenced keratins are expressed in the epidermis (subtype IIe), and two in simple epithelia and mesenchymal cells (subtype IIs). The IIs keratins are both orthologs of human K8. This leaves unsequenced only the trace component S3 of the biochemically established trout keratin catalog. A phylogenetic tree has been constructed from a multiple alignment of the rod domains of the new keratin sequences together with type II sequences from other vertebrates such as shark, zebrafish, and human; lamprey K8 (recently sequenced in our laboratory) has been used as outgroup. This tree suggests, in a highly bootstrap-supported manner, that the teleost IIe keratins diversified independently from the mammalian IIe keratins. In contrast, all the species investigated express K8-like keratins, suggesting that the different IIe branches evolved from K8-like progenitors. The tree also indicates that the published zebrafish sequences represent IIe keratins and that the biochemically identified K8 ortholog in zebrafish has not yet been sequenced.     

Antibodies to different isoforms of the heavy neurofilament protein (NF-H) in normal aging and Alzheimer's disease

Sera of normal controls and of patients with neurological diseases contain antineurofilament antibodies. Recent studies suggest that biochemically and immunologically distinct subclasses of neurofilaments occur in different types of neurons. Alzheimer's disease (AD), the major cause of dementia, is associated with a marked degeneration of brain cholinergic neurons. In the present work we characterized the repertoire and age dependence of antineurofilament antibodies in normal sera and examined whether the degeneration of cholinergic neurons in AD is associated with serum antibodies directed specifically against the neurofilaments of mammalian cholinergic neurons. This was performed by immunoblot assays utilizing neurofilaments from the purely cholinergic bovine ventral root neurons and from the chemically heterogeneous bovine dorsal root neurons. Antibodies to the heavy neurofilament protein NF-H were detected in normal control sera. Their levels were significantly higher in older (aged 70–79) than in younger (aged 40–59) subjects. These antibodies bound similarly to bovine ventral root and dorsal root NF-H and their NF-H specificity was unchanged during aging. In contrast, the levels of IgG in AD sera that are directed against ventral root cholinergic NF-H were higher than those directed against the chemically heterogeneous dorsal root NF-H. Immunoblot experiments utilizing dephosphorylated ventral root and dorsal root NF-H and chymotryptic fragments of these molecules revealed that AD sera contain a repertoire of antimamalian NF-H IgG. A subpopulation of these antibodies binds to phosphorylated epitopes that are specifically enriched in ventral root cholinergic NF-H and that are located on the carboxy terminal domain of this molecule. The level of these anticholinergic NF-H IgG are significantly higher in AD sera than in those of both normal controls and patients with multi-infarct dementia.     

Phosphorylation and dephosphorylation of distinct isoforms of the heavy neurofilament protein NF-H

Summary 1. Previous immunohistochemical studies led to the suggestion that distinctly phosphorylated neurofilament isoforms exist in different types of neurons. We have recently examined this hypothesis by direct biochemical experiments, which revealed that the heavy neurofilament protein NF-H of bovine ventral root cholinergic neurons is more acidic and markedly more phosphorylated than that of bovine dorsal root neurons.2. In the present study we employed this system to study the degree to which distinctly phosphorylated NF-H isoforms differ in the extents to which they can be phosphorylated and dephosphorylatedin vitro. This was performed utilizing alkaline phosphatase and protein kinase PK40^ERK, which is specific to serines of Lys-Ser-Pro (KSP) repeats. The results obtained reveal that:3. The more extensively phosphorylated ventral root NF-H is dephosphorylated more rapidly than dorsal root NF-H.4. Ventral root NF-H and dorsal root NF-H in their native form are both poor substrates of PK40^ERK.5. Following dephosphorylation, ventral root and dorsal root NF-H are phosphorylated extensively and differentially by this kinase. Under these conditions, PK40^ERK catalyzes the incorporation of, respectively, 4.2±1.3 and 2.8±0.6 mol of phosphate per molecule of ventral root NF-H and dorsal root NF-H. The ratio of phosphates incorporated into ventral root NF-H to those incorporated into dorsal root NF-H is 1.46±0.17.6. These findings support the hypothesis that different classes of neurons contain distinctly phosphorylated neurofilaments and show that ventral root and dorsal root neurons are a useful model system for studying the distinct characteristics of neurofilament phosphorylation in different types of neurons.     

Mechanism and evolution of protein dimerization.

We have investigated the mechanism and the evolutionary pathway of protein dimerization through analysis of experimental structures of dimers. We propose that the evolution of dimers may have multiple pathways, including (1) formation of a functional dimer directly without going through an ancestor monomer, (2) formation of a stable monomer as an intermediate followed by mutations of its surface residues, and (3), a domain swapping mechanism, replacing one segment in a monomer by an equivalent segment from an identical chain in the dimer. Some of the dimers which are governed by a domain swapping mechanism may have evolved at an earlier stage of evolution via the second mechanism. Here, we follow the theory that the kinetic pathway reflects the evolutionary pathway. We analyze the structure-kinetics-evolution relationship for a collection of symmetric homodimers classified into three groups: (1) 14 dimers, which were referred to as domain swapping dimers in the literature; (2) nine 2-state dimers, which have no measurable intermediates in equilibrium denaturation; and (3), eight 3-state dimers, which have stable intermediates in equilibrium denaturation. The analysis consists of the following stages: (i) The dimer is divided into two structural units, which have twofold symmetry. Each unit contains a contiguous segment from one polypeptide chain of the dimer, and its complementary contiguous segment from the other chain. (ii) The division is repeated progressively, with different combinations of the two segments in each unit. (iii) The coefficient of compactness is calculated for the units in all divisions. The coefficients obtained for different cuttings of a dimer form a compactness profile. The profile probes the structural organization of the two chains in a dimer and the stability of the monomeric state. We describe the features of the compactness profiles in each of the three dimer groups. The profiles identify the swapping segments in domain swapping dimers, and can usually predict whether a dimer has domain swapping. The kinetics of dimerization indicates that some dimers which have been assigned in the literature as domain swapping cases, dimerize through the 2-state kinetics, rather than through swapping segments of performed monomers. The compactness profiles indicate a wide spectrum in the kinetics of dimerization: dimers having no intermediate stable monomers; dimers having an intermediate with a stable monomer structure; and dimers having an intermediate with a stable structure in part of the monomer. These correspond to the multiple evolutionary pathways for dimer formation. The evolutionary mechanisms proposed here for dimers are applicable to other oligomers as well.     

The beta4 integrin interactor p27(BBP/eIF6) is an essential nuclear matrix protein involved in 60S ribosomal subunit assembly

p27(BBP/eIF6) is an evolutionarily conserved protein that was originally identified as p27(BBP), an interactor of the cytoplasmic domain of integrin beta4 and, independently, as the putative translation initiation factor eIF6. To establish the in vivo function of p27(BBP/eIF6), its topographical distribution was investigated in mammalian cells and the effects of disrupting the corresponding gene was studied in the budding yeast, Saccharomyces cerevisiae. In epithelial cells containing beta4 integrin, p27(BBP/eIF6) is present in the cytoplasm and enriched at hemidesmosomes with a pattern similar to that of beta4 integrin. Surprisingly, in the absence and in the presence of the beta4 integrin subunit, p27(BBP/eIF6) is in the nucleolus and associated with the nuclear matrix. Deletion of the IIH S. cerevisiae gene, encoding the yeast p27(BBP/eIF6) homologue, is lethal, and depletion of the corresponding gene product is associated with a dramatic decrease of the level of free ribosomal 60S subunit. Furthermore, human p27(BBP/eIF6) can rescue the lethal effect of the iihDelta yeast mutation. The data obtained in vivo suggest an evolutionarily conserved function of p27(BBP/eIF6) in ribosome biogenesis or assembly rather than in translation. A further function related to the beta4 integrin subunit may have evolved specifically in higher eukaryotic cells.     

Further similarities between astrocytes and perisinusoidal stellate cells of liver (Ito cells): Colocalization of desmin and glial fibrillary acidic protein in astroglial primary cultures

The colocalization of desmin and glial fibrillary acidic protein (GFAP) in astrocytes was inferred from previous studies demonstrating a unique antigenic composition comprising GFAP, desmin and vimentin in perisinusoidal stellate cells (PSC) of liver which share several features with astrocytes. In the present study the colocalization of GFAP and desmin was investigated by double-immunolabeling experiments in 12-day-old rat astroglial primary cultures with antiserum against GFAP and two commercial monoclonal antibodies against desmin, antibodies of clone DEU-10 and clone DEB-5. These antibodies selectively decorated the perisinusoidal stellate cells (PSC) of liver for which desmin is known to be a marker. The results obtained with astroglial cells demonstrate that both GFAP and desmin are coexpressed in morphologically different types, process-bearing and process-lacking astrocytes. The expression of desmin was apparently more pronounced in process-lacking astrocytes and was considerably lower in process-bearing ones. In process-lacking astrocytes, in contrast to filamentous cytoplasmic staining for GFAP, the immunoreactivity for desmin was non-filamentous and was irregularly spread in the perinuclear cytoplasm of the cells, while in process-bearing astrocytes the pattern of staining for desmin was similar to that of GFAP. The variability in the intensity and pattern of staining for desmin in astrocytes might be due to transitional stages of differentiation for part of the cells. This interpretation was supported by the presence of GFAP in the cells weakly expressing smooth muscle alpha-actin and the absence of GFAP in the cells enriched with microfilaments.     

The Cl effect on photosynthetic oxygen evolution: interaction of Cl with 18-kDa, 24-kDa and 33-kDa proteins

The interaction of Cl⁻ with the extrinsic proteins of 18 kDa, 24 kDa and 33 kDa in the photosynthetic oxygen-evolution complex was studied by comparing spinach photosystem II particles of different protein compositions. The 33-kDa protein decreased the Cl⁻ concentration optimum for oxygen evolution from 150 to 30 mM, and the 24-kDa protein decreased it from 30 to 10 mM. The 18-kDa protein did not change the optimum Cl⁻ concentration, but sustained oxygen evolution at Cl⁻ concentrations lower than 3 mM. The presence of the 24-kDa and 18-kDa proteins, but not each protein alone, markedly suppressed inactivation of oxygen evolution at a very low Cl⁻ concentration and its restoration by readdition of Cl⁻.     

中间纤维与疾病

中间纤维(intermediate filament,IF)与微管、微丝一起组成细胞骨架的蛋白质纤维网络体系。三种骨架纤维中最复杂的是IF,它由最大的基因家族所编码,组成了一个包含73个成员的蛋白大家族。IF除了支架作用还形成复杂的信息平台,并与各种激酶、受体和凋亡蛋白相互作用。目前,已知80多种人类相关疾病包括皮肤起泡、肌肉萎缩症、心肌病、早衰综合征、神经退行性疾病和白内障等与IF有关,且数量仍在增长。其中,IF的变异至少与30多种人类组织特异性疾病有关,在几种神经退行性疾病、肌肉疾病或其他相关疾病还会出现特征性的包涵体。IF可作为细胞类型的标志,其抗体被广泛应用于病理诊断,因此研究这些疾病与IF之间的相互联系、揭示它们的作用机制对全面认识IF在细胞和组织中所起的作用以及对临床疾病的治疗有着重要意义。     

Selective loss of neurofilament expression in Cu/Zn superoxide dismutase (SOD1) linked amyotrophic lateral sclerosis

Neurofilament pathology is a hallmark of sporadic and familial amyotrophic lateral sclerosis (SALS and FALS). The disease mechanisms underlying this pathology are presently unclear, but recent evidence in SALS patients suggest that reductions in neurofilament light subunit (NFL) mRNA may contribute to the death of motor neurones. Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) represent the best-studied cause of FALS, and a number of laboratory models of SOD1-mediated disease exist. Here we have used microdissected lumbar spinal cord motor neurones from human SOD1 FALS patients as well as G93A SOD1 transgenic mice and demonstrated that reduced NFL mRNA levels are seen in both. To probe the molecular mechanisms underpinning these observations, we generated NSC34 motor neurone-like cell lines expressing wild-type and mutant SOD1. NSC34 cells expressing G37R or G93A SOD1 showed selective reductions in NFL and NFM mRNA and protein. These data suggest that NFL mRNA reductions are common to SALS and FALS patients, and that cells and mice expressing mutant SOD1 may enable us to characterize the molecular mechanism(s) responsible for the loss of neurofilament mRNA.     

Primary structure and evolution of calcium-activated neutral protease (CANP)

The amino acid sequences of two subunits (80K and 30K) of calcium-activated neutral protease (CANP) were examined to clarify the structure-function relationship of CANP. The 80K subunit is composed of four clear domains (I–IV from the N-terminus). Domain II is a cysteine proteinase domain homologous to cathepsins B, L, and H. Domain IV is a calcium binding domain with four consecutive EF-hand structures known as typical calcium-binding sites found in calmodulin. The 30K subunit also has a clear domain structure (two domains). The N-terminal domain, a Gly-rich hydrophobic domain, probably determines the location of CANP through association with cellular membrane. The C-terminal domain is a calmodulinlike calcium-binding domain highly homologous to IV in the 80K subunit. The protease activity ascribable to II is regulated by 2 moles of built-in calmodulins, though its precise regulation mechanism is unknown. These results are discussed together with the molecular evolution of CANP on the basis of the gene structures of the two subunits.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Cytoskeletal markers and specific protein production in cells cultured from human first and third trimester placentae

Summary In primary, short-term cultures derived from first and third trimester placentae, 60 to 90 and 70 to 95%, respectively, of the total cell population positively stain for cytokeratin intermediate filaments, typical of epithelial, i.e. trophoblastic cells. The rest of the cells express only vimentin intermediate filaments and thus are of mesenchymal origin. Only the cytokeratin-positive cells express human chorionic gonadotropin (hCG), whereas both the epithelial and the mesenchymal cells stain positively for pregnancy-specific beta-1-glycoprtein (SP₁). Cytokeratin-negative and vimentin-positive cell overgrowth is observed in cultures derived from first and early third trimester placentae. The cells constituting the monolayer thus formed are of fetal origin as evidenced by the expression of Y-body in over 80% of them. The cultured cells synthesize and secrete hCG and SP₁. The activity of these trophoblast-specific functions is inversely proportional to the gestational age of the placenta. Production of specific proteins and expression of intermediate filaments are proposed as criteria for defining the nature and origin of placental cells in primary, short-term cultures.     

Functional divergence in protein (family) sequence evolution

As widely used today to infer function, the homology search is based on the neutral theory that sites of greatest functional significance are under the strongest selective constraints as well as lowest evolutionary rates, and vice versa. Therefore, site-specific rate changes (or altered selective constraints) are related to functional divergence during protein (family) evolution. In this paper, we review our recent work about this issue. We show a great deal of functional information can be obtained from the evolutionary perspective, which can in turn be used to facilitate high throughput functional assays. The emergence of evolutionary functional genomics is also indicated. The related software DIVERGE can be obtained form http://xgu1.zool.iastate.edu.     

Development and characterization of a human cell line from an ovarian mixed mullerian tumor (carcinosarcoma)

Summary A cell line derived from a human ovarian carcinosarcoma was established in tissue culture and in nude mice. Two sublines, LDF and HDF, separated by discontinuous density centrifugation were also established from the parent line JoN. The cloning efficiency of the JoN line was 21%. Morphologic features of adenocarcinoma cells characteristic of the parent JoN cells were retained in the sublines and clones; all lines showed the same karyotype and DNA content (pseudodiploid and pseudotetraploid). Keratin, as demonstrated immunohistochemically, was strongly expressed in the parent line JoN and the xenograft tumor, but not at all in the LDF sublines and only moderately in the HDF sublines. Vimentin, however, was expressed in neither the parent line JoN nor the xenograft tumor, but was present in both sublines. Transglutaminase and plasminogen activator activity was high in the parent line JoN. Neither, sublines nor clones showed the same high enzyme activity as the parent line. It is concluded that this human tumor line JoN is comprised of epithelial cells, capable of multidirectional differentiation.     

Domain tree-based analysis of protein architecture evolution

Understanding the dynamics behind domain architecture evolution is of great importance to unravel the functions of proteins. Complex architectures have been created throughout evolution by rearrangement and duplication events. An interesting question is how many times a particular architecture has been created, a form of convergent evolution or domain architecture reinvention. Previous studies have approached this issue by comparing architectures found in different species. We wanted to achieve a finer-grained analysis by reconstructing protein architectures on complete domain trees. The prevalence of domain architecture reinvention in 96 genomes was investigated with a novel domain tree-based method that uses maximum parsimony for inferring ancestral protein architectures. Domain architectures were taken from Pfam. To ensure robustness, we applied the method to bootstrap trees and only considered results with strong statistical support. We detected multiple origins for 12.4% of the scored architectures. In a much smaller data set, the subset of completely domain-assigned proteins, the figure was 5.6%. These results indicate that domain architecture reinvention is a much more common phenomenon than previously thought. We also determined which domains are most frequent in multiply created architectures and assessed whether specific functions could be attributed to them. However, no strong functional bias was found in architectures with multiple origins.     

Type I keratin cDNAs from the rainbow trout: independent radiation of keratins in fish

Five different type I keratins from a teleost fish, the rainbow trout Oncorhynchus mykiss, have been sequenced by cDNA cloning and identified at the protein level by peptide mass mapping using MALDI-MS. This showed that the entire range of type I keratins detected biochemically in this fish has now been sequenced. Three of the keratins are expressed in the epidermis (subtype Ie), whereas the other two occur in simple epithelia and mesenchymal cells (subtype Is). Among the Is keratins is an ortholog of human K18; the second Is polypeptide is clearly distinct from K18. We raised a new monoclonal antibody (F1F2, subclass IgG1) that specifically recognizes trout Is keratins, with negative reactions on zebrafish. A phylogenetic tree has been constructed from a multiple alignment of the rod domains of the new sequences together with type I sequences from other vertebrates such as shark, zebrafish, and human; a recently sequenced lamprey Is keratin was applied as outgroup. This tree shows one branch defining the K18 orthologs and a second branch containing all other type I keratins (mostly subtype Ie). Within this second branch, the teleost keratins form a separate, highly bootstrap-supported twig. This tree leaves little doubt that the teleost Ie keratins diversified independently from the mammalian Ie keratins.     

Maturation of neurites in mixed cultures of spinal cord neurons and muscle cells from Xenopus laevis embryos followed with antibodies to neurofilament proteins

Dissociated cell cultures of Xeopus laevis embryonic spinal cord have proved useful for studying the differentiation of neuronal ionic channel and membrane properties and for examining the dynamics of microtubules in developing neurons. To examine their usefulness for studying neurofilaments in developing neurites, we prepared similar cultures from stage 22 embryos. Between 3 and 55 h after plating, these cultures were fixed and immunostained with antibodies directed against various epitopes of neurofilament proteins from X. Laevis. These antibodies were specific for nonphosphorylated epitopes of the two low molecular weight Xenopus neurofilament proteins (Xenopus NF-L and the Xenopus neuronal intermediate filament protein, XNIF), both phosphorylated and nonphosphorylated epitopes of the Xenopus middle molecular weight neurofilament protein (NF-M), and a nonphosphorylated epitope of the Xenopus high molecular weight neurofilament protein (NF-H). The emergence of these neurofilament proteins in culture was compared to the time course previously reported for them in Xenopus spinal cord neurons in situ. To facilitate the comparison of times in culture to developmental stages, the age of cultured neurons was converted to an equivalent Nieuwkoop and Faber normal stage using data presented here on the effect of changing temperature on developmental rates of X. laevis. With the exception of the nonphosphorylated epitope of NF-H, which is indicative of the most mature axons found in situ. the emergence of the other neurofilament protein antibody epitopes closely paralleled that previously reported for these antibodies in situ. Thus, with respect to XNIF, NF-M, and NF-L, the neurities of cultured neurons were typical of young embryonic Xenopus laevis spinal cord axons. This system should prove useful for studying both the function of these neurofilament proteins during the early stages of axonal development and the dynamics of their transport. 1994 John Wiley & Sons, Inc.