Cytoplasmic intermediate filament proteins and the nuclear lamins A, B and C share the IFA epitope

The murine monoclonal antibody IFA isolated by Pruss et al. (Cell 27 (1981) 419) reacts with all major proteins of the cytoplasmic intermediate filament family (IF) albeit with different affinities but leaves the nucleus undecorated in standard immunofluorescence microscopy. Here we show that IFA reacts with all three nuclear lamins from rat and man in immunoblotting. This is most easily demonstrated in a cell line in which most cells lack cytoplasmic IFs. Thus the rather minor but ubiquitous 66 kD polypeptides identified by Pruss et al. as IF-associated proteins reflect the lamin triplet. While surprising at first, these results are in agreement with the approximate location of the IFA epitope on IF molecules and the recently discovered sequence homology along the rod domain between lamins A and C and IF proteins. Our results extend this relation to lamin B in spite of its unique behaviour during mitosis.     

Structure of an invertebrate gene encoding cytoplasmic intermediate filament (IF) proteins: implications for the origin and the diversification of IF proteins.

The structure of the single gene encoding the cytoplasmic intermediate filament (IF) proteins in non-neuronal cells of the gastropod Helix aspersa is described. Genomic and cDNA sequences show that the gene is composed of 10 introns and 11 exons, spanning greater than 60 kb of DNA. Alternative RNA processing accounts for two mRNA families which encode two IF proteins differing only in their C-terminal sequence. The intron/exon organization of the Helix rod domain is identical to that of the vertebrate type III IF genes in spite of low overall protein sequence homology and the presence of an additional 42 residues in coil 1b of the invertebrate sequence. Intron position homology extends to the entire coding sequence comprising both the rod and tail domains when the invertebrate IF gene is compared with the nuclear lamin LIII gene of Xenopus laevis presented in the accompanying report of Döring and Stick. In contrast the intron patterns of the tail domains of the invertebrate IF and the lamin genes differ from those of the vertebrate type III genes. The combined data are in line with an evolutionary descent of cytoplasmic IF proteins from a nuclear lamin-like progenitor and suggest a mechanism for this derivation. The unique position of intron 7 in the Helix IF gene indicates that the archetype IF gene arose by the elimination of the nuclear localization sequence due to the recruitment of a novel splice site. The presumptive structural organization of the archetype IF gene allows predictions with respect to the later diversification of metazoan IF genes. Whereas models proposing a direct derivation of neurofilament genes seem unlikely, the earlier speculation of an mRNA transposition mechanism is compatible with current results.     

Cytoplasmic intermediate filament proteins of invertebrates are closer to nuclear lamins than are vertebrate intermediate filament proteins; sequence characterization of two muscle proteins of a nematode.

The giant body muscle cells of the nematode Ascaris lumbricoides show a complex three dimensional array of intermediate filaments (IFs). They contain two proteins, A (71 kd) and B (63 kd), which we now show are able to form homopolymeric filaments in vitro. The complete amino acid sequence of B and 80% of A have been determined. A and B are two homologous proteins with a 55% sequence identity over the rod and tail domains. Sequence comparisons with the only other invertebrate IF protein currently known (Helix pomatia) and with vertebrate IF proteins show that along the coiled-coil rod domain, sequence principles rather than actual sequences are conserved in evolution. Noticeable exceptions are the consensus sequences at the ends of the rod, which probably play a direct role in IF assembly. Like the Helix IF protein the nematode proteins have six extra heptads in the coil 1b segment. These are characteristic of nuclear lamins from vertebrates and invertebrates and are not found in vertebrate IF proteins. Unexpectedly the enhanced homology between lamins and invertebrate IF proteins continues in the tail domains, which in vertebrate IF proteins totally diverge. The sequence alignment necessitates the introduction of a 15 residue deletion in the tail domain of all three invertebrate IF proteins. Its location coincides with the position of the karyophilic signal sequence, which dictates nuclear entry of the lamins. The results provide the first molecular support for the speculation that nuclear lamins and cytoplasmic IF proteins arose in eukaryotic evolution from a common lamin-like predecessor.     

Amino acid sequences and homopolymer-forming ability of the intermediate filament proteins from an invertebrate epithelium.

Intermediate filaments (IF) isolated from the oesophagus epithelium of the snail Helix pomatia contain two polypeptides of mol. wt 66,000 (A) and 52,000 (B), which we have now characterized by in vitro self-assembly studies and by protein sequences. A and B can each form morphologically normal IF and share extended regions of sequence identity. All A-specific sequences seem to locate to an extension of the carboxyl-terminal domain. Although the Helix protein(s) reveal the IF-consensus sequences at the ends of the coiled-coil, the remainder of the rod domain shows conservation of sequence principles rather than extended homology, when compared with any subtype of vertebrate IF proteins. Interestingly, the Helix proteins have the longer coil 1b domain found in nuclear lamins and not in cytoplasmic IF proteins of vertebrates. They lack, however, the karyophilic signal sequence typical for lamins. Obvious implications for IF evolution and structure are discussed.     

Molecular Phylogeny of Metazoan Intermediate Filament Proteins

We have cloned cytoplasmic intermediate filament (IF) proteins from a large number of invertebrate phyla using cDNA probes, the monoclonal antibody IFA, peptide sequence information, and various RT-PCR procedures. Novel IF protein sequences reported here include the urochordata and nine protostomic phyla, i.e., Annelida, Brachiopoda, Chaetognatha, Echiura, Nematomorpha, Nemertea, Platyhelminthes, Phoronida, and Sipuncula. Taken together with the wealth of data on IF proteins of vertebrates and the results on IF proteins of Cephalochordata, Mollusca, Annelida, and Nematoda, two IF prototypes emerge. The L-type, which includes 35 sequences from 11 protostomic phyla, shares with the nuclear lamins the long version of the coil 1b subdomain and, in most cases, a homology segment of some 120 residues in the carboxyterminal tail domain. The S-type, which includes all four subfamilies (types I to IV) of vertebrate IF proteins, lacks 42 residues in the coil 1b subdomain and the carboxyterminal lamin homology segment. Since IF proteins from all three phyla of the chordates have the 42-residue deletion, this deletion arose in a progenitor prior to the divergence of the chordates into the urochordate, cephalochordate, and vertebrate lineages, possibly already at the origin of the deuterostomic branch. Four phyla recently placed into the protostomia on grounds of their 18S rDNA sequences (Brachiopoda, Nemertea, Phoronida, and Platyhelminthes) show IF proteins of the L-type and fit by sequence identity criteria into the lophotrochozoic branch of the protostomia. Received: 2 April 1998 / Accepted: 19 June 1998     

Identification of the region including the epitope for a monoclonal antibody which can neutralize human parvovirus B19.

In this study, we identified a region in the human parvovirus structural protein which involves the neutralization of the virus by a monoclonal antibody and site-specific synthetic peptides. A newly established monoclonal antibody reacted with both viral capsid proteins VP1 and VP2. The epitope was found in six strains of independently isolated human parvovirus B19. The monoclonal antibody could protect colony-forming unit erythroid in human bone marrow cell culture from injury by the virus. The monoclonal antibody reacted with only 1 of 12 peptides that were synthesized according to a predicted amino acid sequence based on nucleotide sequences of the coding region for the structural protein of B19 virus. The sequence recognized by the antibody was a site corresponding to amino acids 328 to 344 from the amino-terminal portion of VP2. This evidence suggests that the epitope of the viral capsid protein is located on the surface of the virus and may be recognized by virus-neutralizing antibodies.     

Modulation of desmin intermediate filament assembly by a monoclonal antibody

We have used a monoclonal antibody against desmin to examine the assembly of intermediate filaments (IF) from their building blocks, the tetrameric protofilaments. The antibody, designated D76, does not cross react with any other IF proteins (Danto, S.I., and D.A. Fischman. 1984. J. Cell Biol. 98:2179-2191). It binds to a region amino-terminal to cys-324 of avian desmin that is resistant to chymotrypsin and trypsin digestion, and in the electron microscope appears to bind to the ends of tetrameric protofilaments. In combination, these findings suggest that the epitope of the antibody resides at the amino-terminal end of the alpha-helical rod domain. Preincubation of desmin protofilaments with an excess of D76 antibodies blocks their subsequent assembly into IF. In the presence of sub-stoichiometric amounts of antibodies, IF are assembled from protofilaments but they are morphologically aberrant in that (a) they are capped by IgG molecules at one or both ends; (b) they are unraveled to varying degree, revealing a characteristic right-handed helical arrangement of sub-filamentous strands of different diameters. The antibody binds only to the ends but not along the length of desmin IF. The most straightforward explanation for this is that the epitope resides in a part of the desmin molecule that becomes buried within the core of the filament upon polymerization and is therefore inaccessible to the antibody.     

Lamins A, B and C share an epitope with the common domain of intermediate filament proteins

Peripheral lamina were isolated from rat liver nuclei and probed with a monoclonal antibody reactive against a common domain of all intermediate filament (IF) proteins. The antibody reacted against lamins A and C as expected from their known sequence homology with IF proteins. Lamin B was also recognized by the antibody. These results indicate that lamin B, despite important biochemical differences vs lamins A and C, also shares sequence homology with IF proteins.     

Localization of a conserved epitope and an azurin-like domain in the H.8 protein of pathogenic Neisseria

The pathogenic neisseriae, Neisseria gonorrhoeae and Neisseria meningitidis, possess an outer membrane protein, H.8, which contains a conserved monoclonal antibody (MAb)-binding epitope in all strains tested. We have cloned and sequenced a meningococcal H.8 gene, and determined the characteristics of the predicted protein. The predicted signal peptide has features characteristic of a prokaryotic lipoprotein. The region at the N-terminal end of the mature protein (39 amino acids) is primarily composed of alanine, glutamate and proline residues arranged in imperfect repeats with the consensus sequence AAEAP. The epitope for H.8 MAb-binding was localized to a 20-amino-acid sequence within this region. The remainder of the predicted amino acid sequence shows extensive homology to azurins, which are small blue copper-binding proteins found in a limited number of species of pathogenic bacteria.     

Microinjection of IFA antibody induces intermediate filament aggregates in epithelial cell lines but perinuclear coils in fibroblast-like lines.

The murine monoclonal IFA antibody recognizes a conserved sequence present in almost all intermediate filament (IF) proteins. When IFA antibody was injected into 13 different primary or established cell lines, striking differences were detected between epithelial and fibroblastic cell lines. In epithelial cells keratin IFs were broken down within 4 h into numerous spheroid aggregates scattered throughout the cytoplasm. Keratin aggregates were first detected in the cytoplasmic periphery. In contrast, in fibroblastic cells, injection of IFA antibody led to the formation of perinuclear coils of vimentin. IFA antibody at a concentration of greater than 1 mg/ml had to be injected to initiate these transitions. When HeLa cells, which contain separate networks of vimentin and keratin filaments, were injected with IFA antibody, vimentin did not form perinuclear coils but was instead found together with keratin in aggregates. Electron micrographs of HeLa cells injected with IFA antibody showed that the aggregates have diameters between 0.5 and 2.6 microns and resembled the keratin aggregates observed in certain mitotic epithelial cells. Although the ultrastructural studies support an association of some aggregates with desmosomes, aggregates were, however, also induced by injection of IFA antibody into human keratinocytes in low calcium medium under conditions where desmosomes were not present.     

Epitope and mimotope for an antibody to the Na, K-ATPase.

The epitope of a monoclonal antibody specific for the alpha 2 isoform of the Na,K-ATPase was determined and its accessibility in native enzyme was examined. Protein fragmentation with N-chlorosuccinimide, formic acid, trypsin, and leucine aminopeptidase indicated binding near the Na,K-ATPase N-terminus but did not unambiguously delineate the extent of the epitope. The ability of the antibody to bind to denatured enzyme made it a good candidate for screening a random peptide library displayed on M13 phage, but the consensus sequence that emerged was not found in the Na,K-ATPase, Full-length cDNA for the Na,K-ATPase was randomly fragmented and cloned into beta-galactosidase to create a lambda gt11 expression library; screening with the antibody yielded a set of overlaps spanning 23 amino acids at the N-terminus. Chimeras of Na,K-ATPase alpha 1 and alpha 2 narrowed down the epitope to 14-19 amino acids. The antibody did not recognize fusion proteins constructed with shorter segments of this epitope. It did recognize a fusion protein containing the M13 library consensus sequence, however, indicating that this sequence, which is rich in proline and hydrophobic amino acids (FPPNFLFPPPP), was a mimotope. The natural epitope, unique to the Na,K-ATPase alpha 2 isoform, was GREYSPAATTAENG. Reconstitution of antibody binding in a foreign context such as M13 PIII protein or beta-galactosidase thus required a relatively large number of amino acids, indicating that antibody mapping approaches must allow for epitopes of significant size. The epitope was accessible in native enzyme and exposed on the cytoplasmic side, documenting the surface exposure of a stretch of amino acids at the N-terminus, where the Na,K-ATPase isoforms differ most.     

A new monoclonal antibody recognizing the amino-terminal consensus sequence of vertebrate intermediate filament proteins

The mouse monoclonal antibody ME 101 raised against human peripherin, an intermediate filament protein (IFP) specific to well defined neuronal populations, recognizes all the major classes of vertebrate IFP in immunoblotting assays. Desmin, GFAP, vimentin, peripherin and the lightest neurofilament protein (NF-L) were cleaved into carboxy- and amino-terminal halves by N-chlorosuccinimide at their unique trytophan residue. Whereas the antibody directed against the epitope common to every IFP (intermediate filament antigen or IFA) and located on the carboxy-terminal end of the rod domain recognizes the carboxy-terminal half, the ME 101 antibody, as the present study illustrates, recognizes specifically the amino-terminal half. From the amino acid sequence data of IFP, it is deduced that the cognate epitope is localized on the amino-terminal part of coil la.     

Aplysia capsulin is localized to egg capsules and egg cordon sheaths and shares sequence homology with Drosophila dec-1 gene products

Differential library screening of an albumen gland cDNA library, Western blot analysis, protein expression, immunolocalization studies, comparative genomics, and secretion assays identified a major Aplysia californica albumen gland protein ('capsulin') that is localized to egg capsules and to the sheaths of the egg cordon. Capsulin shared sequence homology with eggshell proteins encoded by the Drosophila dec-1 gene. The 1790-amino acid A. californica precursor contains 17 repeat sequences that are flanked by basic residue processing sites. The numerous proteolytic processing sites may facilitate the breakdown of capsulin prior to when veliger larvae break out of egg capsules as free-swimming larvae. An Aplysia brasiliana capsulin repeat sequence was 97% identical to its A. californica homolog. Capsulin fragments were not detected in the eluates of egg cordons, suggesting that capsulin is not a candidate water-borne pheromone precursor.     

The H8 antigen of pathogenic Neisseriae

We cloned and sequenced the H8 gene from N. meningitidis FAM18. The predicted amino acid sequence included a consensus lipoprotein signal sequence processing site, consistent with lipid modification that could account for the unusual electrophoretic and solubilization properties of H8. The amino acid sequence was rich in alanine and proline, especially in an imperfectly periodic region near the amino terminus, which encompassed the epitope recognized by available monoclonal antibodies. In a panel of neisserial strains, the presence of DNA homologous to the H8 gene correlated with the expression of an H8 protein. We cloned a gene from N. meningitidis JB515 that was distinct from the H8 gene but encoded a protein also recognized by an anti-H8 monoclonal antibody. Mice were not protected from meningococcemia by passive immunization with such an antibody.     

Monoclonal antibodies as probes for determining the microheterogeneity of the link proteins of cartilage proteoglycan

Monoclonal antibodies were raised against Swarm rat chondrosarcoma link protein 2. Two of the resultant hybridomas (9/30/6-A-1 and 9/30/8-A-4) were used in structural analyses of the link proteins. The 9/30/6-A-1 monoclonal antibody recognized an epitope which was only present on rat chondrosarcoma link protein 2. This epitope was absent in rat chondrosarcoma link protein 3 obtained after trypsin or clostripain treatment of rat chondrosarcoma proteoglycan aggregate, indicating that proteolytic digestion either removed or modified the epitope. Contrasting this, the 9/30/8-A-4 monoclonal antibody recognized an epitope present in link protein(s) 1, 2, or 3 isolated from cartilage of several animal species (rat, bovine, human, and chicken). Rat chondrosarcoma link protein 2 was digested with Staphylococcus aureus V8 protease, and the resulting peptides were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to immunolocation analyses. The 9/30/6-A-1 and 9/30/8-A-4 monoclonal antibodies recognized epitopes in two different halves of the link protein molecule. The 9/30/8-A-4 monoclonal antibody was used to identify proteolytic cleavage peptides common to the individual link proteins (1, 2, or 3) purified from cartilage proteoglycans of several animal species. Digestion of rat chondrosarcoma link protein 2 with endoglycosidase H or alpha-mannosidase increased its electrophoretic mobility to that of link protein 3 and removed or altered the determinant recognized by the 9/30/6-A-1 monoclonal antibody, indicating that a high-mannose oligosaccharide chain was part of the antigenic determinant. The 9/30/8-A-4 monoclonal recognition of epitope was unaffected by endo- or exoglycosidase treatment. Endo- and exoglycosidase treatment of bovine nasal cartilage link proteins also altered their electrophoretic mobility, indicating that high-mannose oligosaccharide structures on the various link proteins (1, 2, or 3) accounted for the microheterogeneity observed in sodium dodecyl sulfate-polyacrylamide gels.     

Epitope mapping of a monoclonal antibody against human thrombin by H/D-exchange mass spectrometry reveals selection of a diverse sequence in a highly conserved protein

The epitope of a monoclonal antibody raised against human thrombin has been determined by hydrogen/deuterium exchange coupled to MALDI mass spectrometry. The antibody epitope was identified as the surface of thrombin that retained deuterium in the presence of the monoclonal antibody compared to control experiments in its absence. Covalent attachment of the antibody to protein G beads and efficient elution of the antigen after deuterium exchange afforded the analysis of all possible epitopes in a single MALDI mass spectrum. The epitope, which was discontinuous, consisting of two peptides close to anion-binding exosite I, was readily identified. The epitope overlapped with, but was not identical to, the thrombomodulin binding site, consistent with inhibition studies. The antibody bound specifically to human thrombin and not to murine or bovine thrombin, although these proteins share 86% identity with the human protein. Interestingly, the epitope turned out to be the more structured of two surface regions in which higher sequence variation between the three species is seen.     

Structural and ligand recognition characteristics of an acetylcholine-binding protein from Aplysia californica

We generated an acetylcholine-binding protein from Aplysia californica by synthesis of a cDNA found in existing data bases and its expression in mammalian cell culture. Its subunit assembly and ligand recognition behavior were compared with the binding protein previously derived from Lymnaea stagnalis. The secreted proteins were purified by elution from columns of attached antibodies directed to the FLAG epitope encoded in the expression construct. Although the sequences of the two proteins from marine and fresh water mollusks exhibit the characteristic features of the extracellular domain of the nicotinic receptor, they only possess 33% amino acid identity. Both assemble as stable pentamers with five binding sites per pentamer, yet they show distinguishing features of stability and sensitivity to epitope tag placement. Both proteins exhibit changes in tryptophan fluorescence upon ligand binding; however, the magnitude of the changes differs greatly. Moreover, certain ligands show marked differences in dissociation constants for the two proteins and can be regarded as distinguishing or signature ligands. Hence, the two soluble proteins from mollusks, which can be studied by a variety of physical methods, become discrete surrogate proteins for the extracellular domains of distinct subtypes of nicotinic acetylcholine receptors.