Nuclear granules recognized by some monoclonal antibodies against intermediate filament protein locate on chromosomes during mitosis

AC54 monoclonal antibody (MAb), an anti-desmin MAb, recognizes both intermediate filaments (IFs) and nuclear granules in BHK21/C13 cells. To investigate nuclear granules, similar MAbs were obtained by using desmin fraction as an antigen. Among them, DSB389 MAb recognized mainly nuclear granules in HeLa and rat liver cells. The nuclear granules in HeLa cells were aligned in arrays, sometimes connected by, or part of, a rope-like structure, and stable against treatment with 0.5% Triton X-100 and 2 M NaCl. They located on or around the chromosomes during mitosis. Essentially the same results were obtained with DSB860 and AC54 MAbs. The distribution of the granules in liver nuclei recognized by DSB389 MAb was similar to that of DNA and was different from that of the nuclear pore complexes. The biological significance of the nuclear granules is discussed.     

NUCLEAR ANTIGENS,AS DNA,OF DSB389 MAB AND SOME OTHER ANTI-DESMIN MONOCLONAL ANTIBODIES

The anti-desmin monoclonal antibodies (MAbs) DSB389, AC54, and DSB860 recognize intermediate filaments (IFs) and nuclear antigens that appear granular, locate around chromosomes, and are insoluble following 0.5% Triton X-100 and cetyltrimethylammonium bromide (CTAB) extraction. Nuclear antigens of the MAbs were searched for in an IFs-deficient clone of SW13 cells. Reactive materials specific to DSB389, AC54, and DSB860 MAbs were trapped at the top of the gel of SDS—agarose—PAGE. The reactivity of the materials disappeared after treatment with DNase I. The reactivity, or trapping of this material at the top of the gel, required previous heat treatment of the sample before application to the gel. The MAbs recognized both single-stranded and double-stranded DNA in ELISA. These results indicate that at least the main nuclear antigens of DSB389, DSB860 and AC54 MAbs are DNA.     

Protein kinase C associates with intermediate filaments and stress fibers.

The subcellular distribution of protein kinase C (PKC) was determined by immunofluorescence using anti-PKC monoclonal antibodies (MAbs). The antibodies used were: (1) 1.9 MAb that is directed against an epitope in the catalytic domain of PKC, (2) 1.3 MAb that recognizes an isozyme of PKC (Mochly-Rosen, D., and Koshland, D. E., 1987, J. Biol. Chem. 262, 2291-2297; Mochly-Rosen, D., et al. 1987 Proc. Natl. Acad. Sci. USA 84, 4660-4664) and (3) MC-2a MAb that is directed against the beta-isozyme of PKC (Usuda, N., et al. 1991, J. Cell Biol. 112, 1241-1247). The cells used in this study were baby hamster kidney cells, vimentin+ and vimentin- clones of SW13 (a human adrenal carcinoma cell line), CEM (a human T cell line), U937 (a histiocytic myeloid cell line), and HL60 (a promyelocytic leukemia cell line). The 1.9 MAb was found to recognize a variety of subcellular components, viz., nucleus (nucleoplasm and nucleolus), cytoplasm, vimentin-type intermediate filaments (IF), stress fibers, and cell membrane. Among these components the beta-isozyme-specific MAbs (1.3 and MC-2a) recognized only the IF network, stress fibers, and edges of the cell membrane. Experiments with vimentin+ and vimentin- mutants of SW13 cells, double indirect immunofluorescence studies with anti-vimentin and anti-PKC antibodies, and drug studies confirmed that the IF network is the predominant cytoskeletal network labeled with all anti-PKC MAbs. Immunoblotting studies with the MC-2a MAb revealed that the observed staining of the IF network was not due to a cross-reaction of the MAb with IF proteins and that the MAb specifically recognizes PKC. These studies, while identifying the diverse cell components to which PKC binds, have demonstrated, for the first time, that PKC associates with the IF network in a variety of cell types. Additionally, the studies have confirmed the studies by others concerning the association of PKC with stress fibers.     

Localization of vimentin and desmin in BHK21/C13 cells and in baby hamster kidney

Specific antibodies against the intermediate filament protein subunits, desmin and vimentin, were used to characterize the fibroblastic tissue culture cell line BHK21/C13 and the cells comprising baby hamster kidney (BHK). The BHK21/C13 cells have previously been shown to contain desmin and vimentin by biochemical techniques. The results from double immunofluorescence analysis show that both immunologically distinct intermediate filament subunit proteins are expressed simultaneously within the same BHK21/C13 cell, and that the filamentous patterns are very similar, if not superimposable even in cells treated with colchicine. There are some cells that may contain vimentin only. Double immunofluorescence on cryostat sections of BHKs and preparations of dissociated kidney cells demonstrate that the cells, most likely smooth muscle, comprising the blood vessel walls contain vimentin and desmin simultaneously. The simultaneous expression of vimentin and desmin is not a phenomenon which is restricted to tissue culture cells. Thus, the simultaneous presence of these two intermediate filament proteins within the BHK21/C13 cell may not be the result of growth in tissue culture.     

Ultrastructural analysis of cytoplasmic intermediate filaments and the nuclear lamina in the mouse plasmacytoma cell line MPC-11 after the induction of vimentin synthesis

We examined cytoplasmic intermediate filaments (IFs) and the nuclear lamina in cells of the mouse plasmacytoma cell line MPC-11 (lacking both IF proteins and lamins A and C) after induction of vimentin synthesis with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) by means of whole-mount immunogold electron microscopy (IEM). The technique of IEM was modified to allow analysis of the cytoskeleton and nuclear lamina of cells grown in suspension culture employing antibodies against vimentin and lamin B. IEM showed that newly synthesized vimentin assembled into IFs which formed anastomosing networks throughout the cytoplasm, radiating primarily from the nucleus. The filaments decorated by gold-conjugated antibodies appeared to make contact with the lipid-depleted nuclear envelope residue either by directly terminating on it or through an indirect link via short fibers of varying diameter. Some filaments terminated on the subunits of the nuclear pore complexes but they did not pass through the pores. In the absence of lamins A and C, lamin B formed a nuclear lamina consisting of a globular-filamentous network anchoring the nuclear pore complexes.     

Immunocytochemical identification of cytoskeletal linkages to smooth muscle cell nuclei and mitochondria

In avian smooth muscle cells, desmin-containing intermediate filaments (IFs) are a prominent component of the cytoskeleton and are readily seen in several domains, including the axial intermediate filament bundle (IFB). Both the nucleus and some of the mitochondria are partly surrounded by elements of the IFB. By using anti-desmin and protein-A-colloidal gold labeling, we have identified intermediate filaments that form linkages with the nuclear envelope and with mitochondria. These linkage regions seem to occupy a proportionately greater part of the mitochondrial surface than of the nuclear envelope. The existence of these linkages in smooth muscle cells is consistent with results that support similar linkages to mitochondria and other cellular structures in various cells that contain either vimentin or keratin IFs. These linkages could functionally restrain or assist in homeostatically restoring organelles to their normal position after the rearrangement that accompanies the substantial shortening of smooth muscle cells.     

Immunohistological localization of desmin, the muscle-type 100 A filament protein, in rat astrocytes and Müller glia

The distribution of glial fibrillary acidic (GFA) protein and desmin was compared in cryostat sections of rat brain, spinal cord, and eye by immunofluorescence and peroxidase-antiperoxidase (PAP) staining. Desmin antisera were raised to antigen purified from chicken gizzard. In rat brain and spinal cord, GFA protein and desmin were selectively localized in astrocytes. Neurons and axons were not stained. The only difference between GFA and desmin antisera was the staining of smooth muscle in small arteries with anti-desmin. It was only in retinal glia that a difference in the localization of the two proteins was apparent. As previously reported, only the glia limitans on the inner surface of the retina was demonstrated with GFA antisera in the normal eye. With anti-desmin Müller fibers spanning the whole thickness of the retina were stained. It is concluded that GFA and desmin form two distinct systems of 100 A filaments in astroglia, as previously reported for GFA and vimentin.     

Copurification of actin and desmin from chicken smooth muscle and their copolymerization in vitro to intermediate filaments

Desmin is a 50,000-mol wt protein that is enriched along with 100-A filaments in chicken gizzard that has been extracted with 1 M KI. Although 1 M KI removes most of the actin from gizzard, a small fraction of this protein remains persistently insoluble, along with desmin. The solubility properties of this actin are the same as for desmin: they are both insoluble in high salt concentrations, but are solubilized at low pH or by agents that dissociate hydrophobic bonds. Desmin may be purified by repeated cycles of solubilization by 1 M acetic acid and subsequent precipitation by neutralization to pH 4. During this process, a constant nonstoichiometric ratio of actin to desmin is attained. Gel filtration on Ultrogel AcA34 in the presence of 0.5% Sarkosyl NL-97 reveals nonmonomeric fractions of actin and desmin that comigrate through the column. Gel filtration on Bio-Gel P300 in the presence of 1 M acetic acid reveals that the majority of desmin is monomeric under these conditions. A small fraction of desmin and all of the actin elute with the excluded volume. When the acetic acid is removed from actin-desmin solutions by dialysis, a gel forms that is composed of filaments with diameters of 120-140 A. These filaments react uniformly with both anti-actin and anti-desmin antiserum. These results suggest that desmin is the major subunit of the muscle 100-A filaments and that it may form nonstoichiometric complexes with actin.     

ON THE ROLE OF MICROTUBULES IN MOVEMENT AND ALIGNMENT OF NUCLEI IN VIRUS-INDUCED SYNCYTIA

Infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes rapid and extensive cell fusion. Time-lapse cinematography shows that when cells fuse, their nuclei migrate straight to the center of the syncytium at rates of 1–2 µ/min. Nuclei are often arranged in long, tightly packed, parallel rows in syncytia derived from the fibroblastic BHK21-F cells. Polarization microscopy shows birefringent material between and parallel to these rows of nuclei, and electron microscopy shows bundles of cytoplasmic microtubules, ~250 A in diameter, and filaments, ~80 A in diameter, parallel to and between the rows of nuclei. Colchicine treatment causes disappearance of microtubules from BHK21-F cells and an apparent increase in the number of 80-A filaments. Although colchicine-treated, SV5-infected cells fuse, their nuclei do not migrate or form rows but remain randomly scattered through the syncytial cytoplasm. Incubation at 4°C does not disrupt microtubules in BHK21-F cells. Rows of nuclei have been isolated from SV5-induced syncytia, and the nuclei in them have been found to be intimately associated with microtubules but not with other cytoplasmic structures. These results suggest that microtubules demarcate cytoplasmic channels through which nuclei migrate and that they may also be involved in the mechanism of nuclear movement.     

Cytoskeletal proteins connecting intermediate filaments to cytoplasmic and nuclear periphery

Intermediate filaments (IFs), together with microtubules and microfilaments build up the cytoskeleton of most eukaryotic cells. Cytoplasmic IFs form a dense filament network radiating from the nucleus and extending to the plasma membrane. The association between the cytoplasmic and nuclear surfaces appears to provide a continuous link important for the organisation of the cytoplasm, for cellular communication, and possibly for the transport into and out of the nucleus. Cytoplasmic IFs approach the nuclear surface, thin fibrils seem to connect the IFs with the nuclear pore complexes and a direct interaction of cytoplasmic IFs with the nuclear lamin B has been observed by in vitro binding studies. However, none of the components that cross-link IFs to the nucleus has been unambiguously identified. Furthermore, if a direct interaction between cytoplasmic IFs and the nuclear lamin B occurs in vivo, the question of how cytoplasmic IFs get access to the nuclear interior remains to be resolved. The association of IFs with the plasma membranes involves different components, some of which are cell type specific. Two specialised complexes in epithelial cells: the desmosome and the hemidesmosome, serve as attachment sites for keratin filaments. Desmoplakin is considered as the cross-linking component of IFs to the desmosomal plaque, whereas BPAG1 (bullous pemphigoid antigen) would cross-link IFs at the hemidesmosomal plaque. In other cell types the modality of how IFs are anchored to the plasma membrane is less well understood. It involves different components such as the spectrin based membrane skeleton, ankyrin, myosin, plectin and certainly many other still unravelled partners. Association between the IFs and cellular membranes plays an important role in determining cell shape and tissue integrity. Thus, the identification and characterisation of the components involved in these interactions will be crucial for understanding the function of intermediate filaments.     

Filensin and phakinin form a novel type of beaded intermediate filaments and coassemble de novo in cultured cells

The fiber cells of the eye lens possess a unique cytoskeletal system known as the "beaded-chain filaments" (BFs). BFs consist of filensin and phakinin, two recently characterized intermediate filament (IF) proteins. To examine the organization and the assembly of these heteropolymeric IFs, we have performed a series of in vitro polymerization studies and transfection experiments. Filaments assembled from purified filensin and phakinin exhibit the characteristic 19-21-nm periodicity seen in many types of IFs upon low angle rotary shadowing. However, quantitative mass-per-length (MPL) measurements indicate that filensin/phakinin filaments comprise two distinct and dissociable components: a core filament and a peripheral filament moiety. Consistent with a nonuniform organization, visualization of unfixed and unstained specimens by scanning transmission electron microscopy (STEM) reveals the the existence of a central filament which is decorated by regularly spaced 12-15-nm-diam beads. Our data suggest that the filamentous core is composed of phakinin, which exhibits a tendency to self-assemble into filament bundles, whereas the beads contain filensin/phakinin hetero-oligomers. Filensin and phakinin copolymerize and form filamentous structures when expressed transiently in cultured cells. Experiments in IF-free SW13 cells reveal that coassembly of the lens-specific proteins in vivo does not require a preexisting IF system. In epithelial MCF-7 cells de novo forming filaments appear to grow from distinct foci and organize as thick, fibrous laminae which line the plasma membrane and the nuclear envelope. However, filament assembly in CHO and SV40-transformed lens- epithelial cells (both of which are fibroblast-like) yields radial networks which codistribute with the endogenous vimentin IFs. These observations document that the filaments formed by lens-specific IF proteins are structurally distinct from ordinary cytoplasmic IFs. Furthermore, the results suggest that the spatial arrangement of filensin/phakinin filaments in vivo is subject to regulation by host- specific factors. These factors may involve cytoskeletal networks (e.g., vimentin IFs) and/or specific sites associated with the cellular membranes.     

Cytoplasmic intermediate filaments are stably associated with nuclear matrices and potentially modulate their DNA-binding function

The tight association of cytoplasmic intermediate filaments (cIFs) with the nucleus and the isolation of crosslinkage products of vimentin with genomic DNA fragments, including nuclear matrix attachment regions (MARs) from proliferating fibroblasts, point to a participation of cIFs in nuclear activities. To test the possibility that cIFs are complementary nuclear matrix elements, the nuclei of a series of cultured cells were subjected to the Li-diiodosalicylate (LIS) extraction protocol developed for the preparation of nuclear matrices and analyzed by immunofluorescence microscopy and immunoblotting with antibodies directed against lamin B and cIF proteins. When nuclei released from hypotonically swollen L929 suspension cells in the presence of digitonin or Triton X-100 were exposed to such strong shearing forces that a considerable number were totally disrupted, a thin, discontinuous layer of vimentin IFs remained tenaciously adhering to still intact nuclei, in apparent coalignment with the nuclear lamina. Even in broken nuclei, the distribution of vimentin followed that of lamin B in areas where the lamina still appeared intact. The same retention of vimentin together with desmin and glial IFs was observed on the nuclei isolated from differentiating C2C12 myoblast and U333 glioma cells, respectively. Nuclei from epithelial cells shed their residual perinuclear IF layers as coherent cytoskeletal ghosts, except for small fractions of vimentin and cytokeratin IFs, which remained in a dot-to cap-like arrangement on the nuclear surface, in apparent codistribution with lamin B. LIS extraction did not bring about a reduction in the cIF protein contents of such nuclei upon their transformation into nuclear matrices. Moreover, in whole mount preparations of mouse embryo fibroblasts, DNA/chromatin emerging from nuclei during LIS extraction mechanically and chemically cleaned the nuclear surface and perinuclear area from loosely anchored cytoplasmic material with the production of broad, IF-free annular spaces, but left substantial fractions of the vimentin IFs in tight association with the nuclear surface. Accordingly, double-immunogold electron microscopy of fixed and permeabilized fibroblasts disclosed a close neighborhood of vimentin IFs and lamin B, with a minimal distance between the nanogold particles of ca. 30 nm. These data indicate an extremely solid interconnection of cIFs with structural elements of the nuclear matrix, and make them, together with their susceptibility to crosslinkage to MARs and other genomic DNA sequences under native conditions, complementary or even integral constituents of the karyoskeleton.     

Desmin filaments are stably associated with the outer nuclear surface in chick myoblasts

Eukaryotic cells have highly organized, interconnected intracellular compartments. The nuclear surface and cytoplasmic cytoskeletal filaments represent compartments involved in such an association. Intermediate filaments are the major cytoskeletal elements in this association. Desmin is a muscle-specific structural protein and one of the earliest known muscle-specific genes to be expressed during cardiac and skeletal muscle development. Desmin filaments have been shown to be associated with the nuclear surface in the myogenic cell line C2C12. Previous studies have revealed that mice lacking desmin develop imperfect muscle, exhibiting the loss of nuclear shape and positioning. In the present work, we have analyzed the association between desmin filaments and the outer nuclear surface in nuclei isolated from pectoral skeletal muscle of chick embryos and in primary chick myogenic cell cultures by using immunofluorescence microscopy, negative staining, immunogold, and transmission electron microscopy. We show that desmin filaments remain firmly attached to the outer nuclear surface after the isolation of nuclei. Furthermore, positive localization of desmin persists after gentle washing of the nuclei with high ionic strength solutions. These data suggest that desmin intermediate filaments are stably and firmly connected to the outer nuclear surface in skeletal muscles cells in vivo and in vitro.     

Three-dimensional distributions of desmin and vimentin in cultured hamster cardiomyocytes using the immunogold deep-etching replica technique

The distributions of desmin and vimentin intermediate filaments in cultured hamster heart cells were examined by immunofluorescent microscopy and an immunogold deep-etching replica technique in combination with electron microscopy. Fluorescent studies showed the overall staining patterns of the myocytes as well as the fibroblasts. Monoclonal antibodies (Da, D₃) to desmin showed punctate staining for the myocytes, while polyclonal desmin (pD) stained in a filamentous pattern. Fibroblasts stained strongly with monoclonal anti-vimentin (Va), but did not stain with the desmin probes. Deep-etched immunogold studies confirmed at the ultrastructural level that monoclonal anti-desmin antibodies stain individual intermediate filaments in an intermittent pattern. Monoclonal (D₃) antibody stained the intermediate filaments heavily and continuously at the cell peripheries, while it stained intermittently in the cell body, similar to the Da monoclonal. Monoclonal anti-vimentin stained only intermediate filaments in fibroblasts. Our studies show a heterogeneity of staining within the cultured heart cells when various anti-desmin and anti-vimentin antibodies are used.     

Early appearance of desmin, the muscle-type intermediate filament protein, in the rat embryo

Antisera raised to desmin, the protein subunit of muscle-type intermediate filaments (IFs), were used to study by indirect immunofluorescence and immunoperoxidase procedures the early development of skeletal muscle in the rat embryo. The specificity of the antisera (Dahl D, Bignami A: J Histochem Cytochem 30:207, 1982) was confirmed by immune blotting on chicken gizzard extracts and purified antigen. Desmin-positive cells were first observed on day 12 by immunofluorescence and on day 13 by the immunoperoxidase procedure. Desmin immunoreactivity was not found in caudal somites in which the dermatome was present, i.e., somites where the dorso-lateral part had maintained its definite boundaries and epithelioid characteristics. Desmin-positive cells were observed within the myotome of cranial somites where the dermatome had disappeared. Compared to day 13, desmin-positive cells had extended ventrally on day 14, while on day 15, they were found in the skeletal musculature of the trunk and the limbs.     

Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms

Dysfunction of plectin, a 500-kD cytolinker protein, leads to skin blistering and muscular dystrophy. Using conditional gene targeting in mice, we show that plectin deficiency results in progressive degenerative alterations in striated muscle, including aggregation and partial loss of intermediate filament (IF) networks, detachment of the contractile apparatus from the sarcolemma, profound changes in myofiber costameric cytoarchitecture, and decreased mitochondrial number and function. Analysis of newly generated plectin isoform-specific knockout mouse models revealed that IF aggregates accumulate in distinct cytoplasmic compartments, depending on which isoform is missing. Our data show that two major plectin isoforms expressed in muscle, plectin 1d and 1f, integrate fibers by specifically targeting and linking desmin IFs to Z-disks and costameres, whereas plectin 1b establishes a linkage to mitochondria. Furthermore, disruption of Z-disk and costamere linkages leads to the pathological condition of epidermolysis bullosa with muscular dystrophy. Our findings establish plectin as the major organizer of desmin IFs in myofibers and provide new insights into plectin- and desmin-related muscular dystrophies.     

Arrangement of desmin intermediate filaments in smooth muscle cells as shown by high-resolution immunocytochemistry

To gain additional information about the arrangement of intermediate filaments (IF) in normal smooth muscle, fresh avian gizzard was processed for immunoelectron microscopy. The protein A-gold immunocytochemical technique was applied for the localization of desmin antigenic sites. Desmin-containing IFs were located in an axial bundle that partially surrounds the nucleus and were associated with numerous mitochondria near the poles of the nucleus. The bundle probably extends the length of the cell. Antibody labeling also showed concentrations of IF around and between cytoplasmic dense bodies (CDB) and also between CDB and membrane-associated dense bodies (MADB). The relationship between the axial bundle and the nucleus and associated mitochondria suggests that the bundle may support and define the position of these organelles in the cell. A fraying or branching of the bundle may integrate the bundle into the remaining cytoskeletal network of the cell.     

Presence of a protein immunologically related to lamin B in the postsynaptic membrane of Torpedo marmorata electrocyte

The Torpedo electrocyte is a flattened syncytium derived from skeletal muscle, characterized by two functionally distinct plasma membrane domains. The electrocyte is filled up with a transversal network of intermediate filaments (IF) of desmin which contact in an end-on fashion both sides of the cell. In this work, we show that polyclonal antibodies specific for lamin B recognizes a component of the plasma membrane of Torpedo electrocyte. This protein which thus shares epitopes with lamin B has a relative molecular mass of 54 kD, an acidic IP of 5.4. It is localized exclusively on the cytoplasmic side of the innervated membrane of the electrocyte at sites of IF-membrane contacts. Since our previous work showed that the noninnervated membrane contains ankyrin (Kordeli, E., J. Cartaud, H. O. Nghiem, L. A. Pradel, C. Dubreuil, D. Paulin, and J.-P. Changeux. 1986. J. Cell Biol. 102:748-761), the present results suggest that desmin filaments may be anchored via the 54-kD protein to the innervated membrane and via ankyrin to the noninnervated membrane. These findings would represent an extension of the model proposed by Georgatos and Blobel (Georgatos, S. D., and G. Blobel. 1987a. J. Cell Biol. 105:105-115) in which type III intermediate size filaments are vectorially inserted to plasma and nuclear membranes by ankyrin and lamin B, respectively.     

Monoclonal antibodies as probes to examine serotype-specific and cross-reactive epitopes of lipopolysaccharides from serotypes O2, O5, and O16 of Pseudomonas aeruginosa.

Serotypes O2, O5, and O16 of Pseudomonas aeruginosa are chemically related, and the O antigens of their lipopolysaccharides share a similar trisaccharide repeat backbone structure. Serotype-specific monoclonal antibodies (MAbs) MF71-3, MF15-4, and MF47-4 against the O2, O5, and O16 serotypes, respectively, were isolated. MAb 18-19, which is cross-reactive with all strains of this chemically related serogroup, was also produced. When column chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated lipopolysaccharide (LPS) samples from each of the serotypes were probed with the MAbs in Western immunoblots, each of the serotype-specific MAbs interacted only with high-molecular-weight bands of the homologous LPS, with a minimum O-antigen chain length of at least 6 to 10 repeats. In contrast, cross-reactive MAb 18-19 was shown to interact in Western immunoblots with the entire LPS banding pattern except the fastest-running band, which lacks O antigen. Chemical modification of P. aeruginosa LPS by alkali treatment and carboxyl reduction abolished reactions between LPS and MAb 18-19, while reactions of modified LPS with serotype-specific MAbs were not affected. Therefore, cross-reactive MAb 18-19 likely recognizes the chemical backbone structure of the O repeat that is common to all three serotypes of the O2-O5-O16 group, while the O-specific MAbs appeared to recognize LPS epitopes that could be presented when 6 to 10 or more O-antigen repeat units are present on the LPS molecule. Thus, the O-specific LPS epitopes likely involve unique chemical structures, glycosidic linkages, and some order of folding of the O side chains.