Rod-like Domain Research Articles

Isolation and characterization of a genomic clone from the murine utrophin locus.

Dystrophin is a large 427-kDa cytoskeletal muscle protein localized to the inner surface of the sarcolemma (Hoffman et al. 1987; Koenig et al. 1988; Minetti et al. 1992). Sequence studies have shown that dystrophin consists of four functionally distinct domains--an N-terminal a-actin binding domain, a central rod-like triple helical repeat domain, a cysteine-rich domain, and a C-terminal domain (Koenig et al. 1988). The C-terminal domain appears to interact with an oligomeric complex of glycoproteins in the plasma membrane (Campbell and Kahl 1989), and it has been proposed that this interaction is responsible for anchoring dystrophin to the sarcolemma. However, some Duchenne muscular dystrophy patients who carry C-terminal deletions in dystrophin demonstrate correct localization of the truncated dystrophin at the sarcolemma (Helliwell et al. 1992). Nevertheless, absence of dystrophin leads to a reduction in dystrophinassociated glycoproteins, and, furthermore, it has been reported recently that a deficiency of one of the dystrophin-associated glycoproteins may be involved in severe childhood autosomal recessive muscular dystrophy (Matsumura et al. 1992). An autosomal homolog to dystrophin (Love et al. 1989; Tinsley et al. 1992), named utrophin, encodes a functionally distinct protein that shows homology to dystrophin over the entire coding region. However, particularly strong conservation is found in those C-terminal domains that are presumed to be involved with membrane association in dystrophin. In human the utrophin gene maps to Chromosome (Chr) 6 (Love et al. 1989), and in mouse, with a human utrophin cDNA, a homologous gene (Dmdl) has been mapped

Mapping of nidogen binding sites for collagen type IV, heparan sulfate proteoglycan, and zinc

Recombinant nidogen fragments comprising the globular domains G1 plus G2, the rod-like domain, and the rod connected to the globe G3 were prepared from the culture media of transfected human cell clones. In addition, domains G1 and G2 were separated from each other after cleavage with chymotrypsin. The purified fragments were characterized by N-terminal sequences, electrophoresis, electron microscopy, and radioimmunoassays and the cell clones by Northern hybridization. Transfection with a construct comprising a large part of domain G3 showed high mRNA levels but no secreted protein, indicating a protein folding problem. All these fragments were used as soluble and/or immobilized ligands in binding assays. This demonstrated major binding sites on domain G2 for collagen IV and heparan sulfate proteoglycan. Affinity chromatography on zinc- and cobalt-loaded columns showed binding of domains G2 and G3 and the rod. Protein binding, but not metal binding, was abolished by reduction and alkylation of nidogen. This allowed for the isolation of several zinc-binding tryptic peptides, four from G2, two from the rod, and one from the G3 domain. Most of these short peptides contained several histidines that are likely to mediate binding. Zinc inhibited efficiently G3-mediated nidogen binding to laminin at 4 degrees C (IC50 approximately 5 microM) but less at higher temperatures. Similarly, zinc inhibited binding to collagen IV and proteoglycan at low temperatures but not at high (37 degrees C) temperatures. This indicates a complex modulation of nidogen binding to other basement membrane proteins by some, but not all, transition metals. Whether the particularly striking effects shown for zinc are of biological relevance remains to be established.

A case of Duchenne muscular dystrophy with truncated dystrophin: Significance of a cysteine-rich domain for functional expression of dystrophin protein

A Duchenne muscular dystrophy case showed truncated dystrophin (320 kDa) with an isoelectric point slightly shifted towards a more alkaline pH. From the polymerase chain reaction and immunochemical analysis data, the expressed dystrophin protein was predicted to lack the portion comprising the tail of the rod-like domain, the cysteine-rich domain, and the head of the C-terminal domain. These results indicated the functional importance of the cysteine-rich domain in the dystrophin protein.

Ascidian entactin/nidogen. Implication of evolution by shuffling two kinds of cysteine-rich motifs.

Entactin/nidogen, a major component of the basement membrane, has a domain structure comprising three globular domains, and thread-like and rod-like domains connecting them. It contains six epidermal-growth-factor-(EGF)-like motifs and one thyroglobulin-like motif. In the present study, ascidian entactin/nidogen has been identified by a monoclonal antibody technique. We prepared anti-(ascidian entactin/nidogen)IgG, named anti-AsEnt1, then cloned the cDNA of ascidian entactin/nidogen using anti-AsEnt1 as a probe, and determined its entire sequence. Mainly because the deduced amino acid sequence exhibited high similarity to mouse entactin and human nidogen, and because the antigen localized in basement membrane of ascidian body-wall muscle, we have concluded that the antigen anti-AsEnt1 corresponds to the ascidian entactin/nidogen homologue. The deduced amino acid sequence of ascidian entactin/nidogen clearly showed that the ascidian homologue also has a domain structure. However, the ascidian homologue lacked the thread-like domain, and the rod-like domain differed from that of mouse entactin in composition, consisting of two kinds of cysteine-rich motifs, that is, the EGF-like motif and the thyroglobulin-like motif. These results suggest that entactin/nidogen have evolved by modifying the domains, especially by shuffling the two kinds of cysteine-rich motifs.

A dystrophin-immunoreactive protein in mammalian brain.

Dystrophin is expressed only in muscle and brain, but is absent from all tissues of the adult mdx mouse, a mutant with a single base substitution in the dystrophin gene. The brains of both normal and mdx mice contain a protein of approximately 230 kDa that is recognised by anti-dystrophin antibodies raised to the N-terminal region of the rod-like domain. Although the N-terminal and central rod regions of dystrophin share structural homologies with spectrin, the 230-kDa protein represents neither of the presently described forms of brain spectrin by a variety of criteria (molecular weight, cerebellar localisation, and developmental regulation) and is distinct from the product of the dystrophin gene. Studies of mdx and normal mouse brain show different postnatal developmental regulation of the 230-kDa dystrophin-immunoreactive protein.

Nidogen mediates the formation of ternary complexes of basement membrane components

Using a recombinant nidogen we have probed the calcium binding potential of various nidogen domains, examined the binding of nidogen to various basement membrane proteins and assessed the ability of nidogen to mediate the formation of ternary complexes between laminin and heparan sulfate proteoglycan and collagen IV and laminin. The results of these experiments indicate that the Ca+2 binding is on the rod-like domain with additional binding observed on the N-terminal G1 domain. With regard to the role of nidogen in mediating complex formation among basement membrane components it was demonstrated that nidogen effectively promotes the formation of a ternary complex between laminin and collagen IV, with both of these components interacting independently with nidogen. Similarly, nidogen mediates a ternary complex formation between laminin and proteoglycan. Interestingly, the interaction between proteoglycan and nidogen is through the protein core of the proteoglycan. We have localized the major interaction sites on nidogen with the proteoglycan core and collagen IV to a region on the globular G2 domain while the C-terminal globe G3 binds to laminin. Ca+2 binding does not appear to be important in either of the binary or ternary complex formations. The data reported allow us to hypothesize that, via the multiple interactions of nidogen with other basement membrane components, nidogen plays a crucial structural role in basement membrane organization and stabilization.

Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV.

Recombinant mouse nidogen and two fragments were produced in mammalian cells and purified from culture medium without resorting to denaturing conditions. The truncated products were fragments Nd-I (positions 1-905) comprising the N-terminal globule and rod-like domain and Nd-II corresponding mainly to the C-terminal globule (position 906-1217). Recombinant nidogen was indistinguishable from authentic nidogen obtained by guanidine dissociation from tumor tissue with respect to size, N-terminal sequence, CD spectra and immunochemical properties. They differed in protease stability and shape indicating that the N-terminal domain of the more native, recombinant protein consists of two globules connected by a flexible segment. This established a new model for the shape of nidogen consisting of three globes of variable mass (31-56 kDa) connected by either a rod-like or a thin segment. Recombinant nidogen formed stable complexes (Kd less than or equal to 1 nM) with laminin and collagen IV in binding assays with soluble and immobilized ligands and as shown by electron microscopy. Inhibition assays demonstrated different binding sites on nidogen for both ligands with different specificities. This was confirmed in studies with fragment Nd-I binding to collagen IV and fragment Nd-II binding to laminin fragment P1. In addition, recombinant nidogen but not Nd-I was able to bridge between laminin or P1 and collagen IV. Formation of such ternary complexes implicates a similar role for nidogen in the supramolecular organization of basement membranes.

Cell adhesion, spreading and neurite stimulation by laminin fragment E8 depends on maintenance of secondary and tertiary structure in its rod and globular domain

The cell adhesion, spreading and neurite-promoting properties of mouse tumor laminin fragment E8, which contains major site(s) responsible for laminin-cell interactions, were probed by proteolytic degradation, denaturation, synthetic peptides and antibody inhibition. Removal of more than half of the N-terminal portion contributing to the rod-like domain did not effect cell attachment or spreading although neurite-promoting activity was reduced. More extensive degradation of the rod or of the globular domains of E8, or separation of the globule from the rod, also resulted in loss of cell spreading activity although weak attachment was found to an A chain subfragment comprising the globular domain and a short piece of the rod. Exposure of E8 to increasing concentrations of dissociating agents produce an apparently reversible denaturation but an irreversible loss of both attachment and neurite-promoting activities, as did reduction and alkylation of disulfide bonds in the globular domain. Although cell adhesion and spreading were blocked by antibodies to an alpha 6 integrin subunit, neurite outgrowth was unaffected, indicating two distinct receptors for these two activities. Furthermore, a synthetic peptide, the sequence of which is found in the vicinity of adhesion and neurite-promoting sites and previously implicated in neurite growth and cell attachment activities, was found to be inactive. These results indicate that the major cell attachment and neurite-promoting sites of laminin are distinct although both require the native conformation of parts of the rod and the terminal globular domain of the long arm of laminin.

Evidence for a specific mechanism of laminin assembly

The specificity of laminin chain assembly was investigated using fragments E8 and C8-9, derived from the long arm of the molecule, whose rod-like domain consists of the alpha-helical regions of the A, B1 and B2 chains. Urea-induced chain separation and unfolding were monitored by transverse urea/polyacrylamide gel electrophoresis (PAGE) and circular dichroism. Separation of the A and disulphide-linked B1-B2 chains occurred at 3.5-4.0 M urea and by 7.0 M urea all residual alpha-helicity was lost. Removal of urea by dialysis resulted in high recoveries (87-100%) of renatured protein which in its apparent molecular mass, alpha-helix content, chain composition, degree of association and ultrastructural appearance was indistinguishable from native E8. Reduction or reduction and alkylation of the chains did not lead to a decrease in their ability to reassemble specifically. Reformation of the single interchain disulphide, linking the B1 and B2 chains, clearly demonstrates that these chains are correctly aligned in parallel and in register in E8 renatured from its reduced chains. Renaturation of E8 from its reduced and alkylated chains precludes a role for disulphide formation in determining chain alignment but suggests rather than it is involved in the stabilisation of the correctly assembled molecule. These results, together with recent sequence data, provide evidence for the interaction of the alpha-helical regions of the A, B1 and B2 chains in the formation of a triple coiled-coil within the long arm of the molecule. The highly specific nature of this interaction suggests that it is the mechanism by which laminin is assembled in vivo.

The binding of heparin to type IV collagen: domain specificity with identification of peptide sequences from the α1(IV) and α2(IV) which preferentially bind heparin

Three distinctive heparin-binding sites were observed in type IV collagen by the use of rotary shadowing: in the NC1 domain and at distances 100 and 300 nm from the NC1 domain. Scatchard analysis indicated different affinities for these sites. Electron microscopic analysis of heparin-type IV collagen interaction with increasing salt concentrations showed the different affinities to be NC1 greater than 100 nm greater than 300 nm. The NC1 domain bound specifically to chondroitin/dermatan sulfate side chains as well. This binding was observed at the electron microscope and in solid-phase binding assays (where chondroitin sulfate could compete for the binding of [3H]heparin to NC1-coated substrata). The triple helix-rich, rod-like domain of type IV collagen did not bind to chondroitin/dermatan sulfate side chains. In solid-phase binding assays only heparin could compete for the binding of [3H]heparin to this domain. In order to more precisely map potential heparin-binding sites in type IV collagen, we chemically synthesized 17 arginine- and lysine-containing peptides from the alpha 1(IV) and alpha 2(IV) chains. Three peptides from the known sequence of the alpha 1(IV) and alpha 2(IV) chains were shown to specifically bind heparin: peptide Hep-I (TAGSCLRKFSTM), from the alpha 1(NC1) chain, peptide Hep-II (LAGSCLARFSTM), a peptide corresponding to the same sequence in peptide Hep-I from the alpha 2 (NC1) chain, and peptide Hep-III (GEFYFDLRLKGDK) which contained an interruption of the triple helical sequence of the alpha 1(IV) chain at about 300 nm from the NC1 domain, were demonstrated to bind heparin in solid-phase binding assays and compete for the binding of [3H]heparin to type IV collagen-coated substrata. Therefore, each of these peptides may represent a potential heparin-binding site in type IV collagen. The mapping of the binding of heparin or related structures, such as heparan sulfate proteoglycan, to specific sequences of type IV collagen could help the understanding of several structural and functional properties of this basement membrane protein as well as interactions with other basement membrane and/or cell surface-associated macromolecules.

Amino acid sequence of mouse nidogen, a multidomain basement membrane protein with binding activity for laminin, collagen IV and cells.

The whole amino acid sequence of nidogen was deduced from cDNA clones isolated from expression libraries and confirmed to approximately 50% by Edman degradation of peptides. The protein consists of some 1217 amino acid residues and a 28-residue signal peptide. The data support a previously proposed dumb-bell model of nidogen by demonstrating a large N-terminal globular domain (641 residues), five EGF-like repeats constituting the rod-like domain (248 residues) and a smaller C-terminal globule (328 residues). Two more EGF-like repeats interrupt the N-terminal and terminate the C-terminal sequences. Weak sequence homologies (25%) were detected between some regions of nidogen, the LDL receptor, thyroglobulin and the EGF precursor. Nidogen contains two consensus sequences for tyrosine sulfation and for asparagine beta-hydroxylation, two N-linked carbohydrate acceptor sites and, within one of the EGF-like repeats an Arg-Gly-Asp sequence. The latter was shown to be functional in cell attachment to nidogen. Binding sites for laminin and collagen IV are present on the C-terminal globule but not yet precisely localized.

Characterization of proteolytic fragments of the laminin‐nidogen complex and their activity in ligand‐binding assays

Some 12 new nidogen and laminin fragments were purified from elastase, thrombin and trypsin digests and characterized by their sizes (22 kDa to greater than 300 kDa), subunit patterns on electrophoresis, partial amino acid sequences, content of specific epitopes and their binding to laminin or nidogen structures in radioligand assays. This permitted the various fragments to be ordered along the dumbbell-shaped structure of nidogen and to compare them with previously described nidogen fragments arising by endogenous proteolysis. Two nidogen fragments (E-50, E-90; 50 kDa and 90 kDa) remain associated with a large laminin fragment in elastase digests of the complex and could be dissociated with 2 M guanidine.HCl. Recombination studies demonstrated Kd = 10-20 nM for this interaction. Nidogen fragments devoid of binding activity included the tryptic peptide T-40 (40 kDa) corresponding to the rod-like domain and several larger fragments extending more to the N-terminus of nidogen. An N-terminal thrombin fragment of about 50 kDa was also inactive. Together the data show a lack of laminin binding to the N-terminal globule and rod of nidogen and provide indirect evidence that this activity is located within or close to its C-terminal globular domain. Nidogen-binding structures of laminin were obtained as two large fragments (greater than 300 kDa), P1X and E1X. They correspond to the short arm structure of laminin with one (E1X) or two (P1X) arms decreased in size to the inner rod-like segment. Shortening in E1X is mainly due to the B1 chain segment including the central globular domain which was identified as a new laminin fragment E10. Binding of E1X and P1X to nidogen was comparable to that of laminin while much lower activity was found for other laminin fragments. A 10-fold lower binding potential was also observed for the laminin-nidogen complex whose structure can now be defined in more precise molecular terms.

Three-dimensional reconstruction of maltoporin from electron microscopy and image processing.

Two dimensional crystals of maltoporin (or phage lambda receptor) were obtained by reconstitution of purified maltoporin trimers and Escherichia coli phospholipids by detergent dialysis. Two different trimer packing forms were observed. One was hexagonal (a = 7.8 nm) and one rectangular (a = 7.8 nm, b = 13.6 nm). In this paper we describe the three-dimensional structure of maltoporin, deduced from the study of the rectangular form by electron microscopy and image processing. At a resolution of approximately 2.5 nm, maltoporin trimers form aqueous channel triplets which appear to merge into a single outlet at the periplasmic surface of the outer membrane. The pore defined by maltoporin has a similar structure to that outlined by the matrix protein. From the results of functional studies by conductance measurement, it is concluded that the three channels defined by maltoporin act, contrary to those formed by the porin (OmpF protein), as a single conducting unit. A tentative outline of the maltoporin promoter is given. Maltoporin appears to be constituted by three different domains: a major rod-like domain spanning the membrane, a minor domain located near the periplasmic surface of the membrane and finally a central domain responsible for the splitting of the channel.

Extended and globular protein domains in cartilage proteoglycans.

Electron microscopy after rotary shadowing and negative staining of the large chondroitin sulphate proteoglycan from rat chondrosarcoma, bovine nasal cartilage and pig laryngeal cartilage demonstrated a unique multidomain structure for the protein core. A main characteristic is a pair of globular domains (diameter 6-8 nm), one of which forms the N-terminal hyaluronate-binding region. They are connected by a 25 nm-long rod-like domain of limited flexibility. This segment is continued by a 280 nm-long polypeptide strand containing most chondroitin sulphate chains (average length 40 nm) in a brush-like array and is terminated by a small C-terminal globular domain. The core protein showed a variable extent of degradation, including the loss of the C-terminal globular domain and sections of variable length of the chondroitin sulphate-bearing strand. The high abundance (30-50%) of the C-terminal domain in some extracted proteoglycan preparations indicated that this structure is present in the cartilage matrix rather than being a precursor-specific segment. It may contain the hepatolectin-like segment deduced from cDNA sequences corresponding to the 3'-end of protein core mRNA [Doege, Fernandez, Hassell, Sasaki & Yamada (1986) J. Biol. Chem. 261, 8108-8111; Sai, Tanaka, Kosher & Tanzer (1986) Proc. Natl. Acad. Sci. 83, 5081-5085; Oldberg, Antonsson & Heinegård (1987) Biochem. J. 243, 255-259].

Molecular structure of short-chain (SC) cartilage collagen by electron microscopy

We have recently observed that aged and/or hypertrophying chondrocytes in culture synthesize a small collagen molecule termed short-chain (SC) collagen. Our previous biochemical studies have suggested that this molecule is slightly less than half the length of "typical" interstitial collagens and should have both a helical, collagenous domain and a nonhelical, globular one. In the present study we have examined the structure of this molecule by electron microscopy of rotary-shadowed preparations and segment-long-spacing crystallites. Rotary-shadowed SC collagen molecules appear as rods with a length of 132 nm and a knob at one end. Preparations of native molecules that have been treated by limited pepsin digestion show only the rod-like domain. These results are consistent with the rod-like domain having the molecular structure of a collagen helix, which is refractory to pepsin digestion, and the knob representing a globular, nonhelical domain. Segment-long-spacing crystallites of pepsin-digested molecules confirm the length of the helical domain to be 132 nm. Positively stained crystallites show a banding pattern different from other collagens.

Troponin and its interactions with tropomyosin: An electron microscope study

Some new features of the troponin complex have been revealed by electron microscope study of rotary shadowed molecules. Our results demonstrate that the troponin complex has both a globular and a rod-like domain. The length of the entire complex is ~265 Å and that of the tail is ~ 160 Å. Isolated troponin T has a shape and dimensions that correspond closely to those of the tail, so that the troponin I and C subunits would comprise most of the globular region of the complex. Native and reconstituted troponin-tropomyosin complexes have also been visualized and show the globular portion of troponin bound at regular intervals along the tropomyosin filaments. These electron microscope results, together with recent biochemical studies, suggest that troponin subunits C and I, and part of subunit T bind near Cys190 of tropomyosin, about one-third of the way along the molecule, with the rest of subunit T extending toward the COOH terminus. This arrangement implies that tropomyosin filaments lie on the actin helix with their COOH termini toward the Z-line. The shape of the complex suggests that troponin may interact with tropomyosin over a considerable portion of its length, and may therefore be important in the dynamics of the switching process.

Collagen self-assembly in vitro. Differentiating specific telopeptide-dependent interactions using selective enzyme modification and the addition of free amino telopeptide.

The thermally induced in vitro self-assembly of collagen molecules to form active fibrils illustrates that collagen molecules themselves contain all of the structural information necessary for assembly. The molecule contains three structural domains, the NH2 and carboxyl-terminal extra helical regions (the telopeptides) and the major triple helical rod-like domain. Proteolytic removal of the short telopeptide domains drastically alters the in vitro self-assembly process. We have examined the specific contributions of each telopeptide to the initiation ("nucleation") and growth stages of self-assembly in collagens modified by selective proteinase treatment and by isolating a peptide containing the amino telopeptide and adding this to both normal and proteinase-modified collagen self-assembly systems. Pronase-modified collagen, devoid of both telopeptides, initiated self-assembly very poorly. Addition of small amounts of intact collagen accelerated the rate of nucleation of pronase-modified collagen. Addition of carboxypeptidase-modified collagen also accelerated the nucleation of pronase-modified collagen, suggesting that the remaining amino telopeptide was involved in nucleation. This was confirmed by isolating the cyanogen bromide fragment of the alpha 1(I) subunit containing the amino telopeptide and finding that it specifically accelerated the nucleation of intact pepsin- and pronase-modified to collagens. The amino telopeptide appears to bind to a specific region within the collagen triple helical domain. The isolated peptide requires thermal pretreatment to be active; hence, this interaction must involve a unique telopeptide conformation. This behavior is compatible with the recent model (Helseth, D. L., Jr., Lechner, J. H., and Veis, A. (1979) Biopolymers 18, 3005-3014) proposed for the conformation of the amino telopeptide and its interaction with a helical receptor site as a step in nucleation. Comparison of the behavior of leucine aminopeptidase- and carboxypeptidase-modified collagens suggests that the carboxyl telopeptide has its major role in the growth stages of self-assembly.

Electron microscopy of sym-SBS block polymers

Binary ABA block copolymers where the central block is elastomeric possess a range of characteristic textures when observed as osmium tetroxide stained thin films (40 nm) in the electron microscope. A study of low styrene content samples (∼25%) prepared from dilute solution indicated three basic morphologies: an irregular phase structure, arrays of linear and hexagonally-packed circular styrene domains. The observed texture was a function of rate of preparation, decreasing rates resulting in greater domain ordering. Domains varied in size from 10 to 24 nm with corresponding spacings in the range 10–45 nm. Grain structure was only clearly observed in arrays of circular domains but quasi-spherulitic patterns occurred in linear morphologies. Coherent and random multilayers were also observed, in some cases giving rise to Moiré fringes. Comparison of observed and calculated area fractions from dilute solution characterization implied a rod-like domain structure, although significant errors were introduced by strains in the thin films.