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D A Parry

Publications and source records attributed to D A Parry.

At least 55 records · Page 3Linked to original sources
PubMed1996

Expression of extracellular matrix macromolecules around demineralized freeze-dried bone allografts.

In the present study histochemical techniques were used to identify specific macromolecular components of the extracellular matrix associated with the tissue reaction to demineralized freeze-dried bone allografts (DFDBA) placed under barrier membranes for ridge augmentation. Small biopsies were obtained from tissues underneath the membranes at various times after placement of the DFDBA and processed for routine immunohistochemistry. Sections were stained with antibodies to osteocalcin, collagen type I, collagen type III, decorin, and biglycan. Non-immune serum, irrelevant antibodies, and omission of the primary antibodies served as negative controls. Histologic examination of the biopsies revealed allograft particles surrounded by well-formed fibrous connective tissue with little or no evidence of new bone formation. Vital autogenous bone fragments were present in the peripheral portions of the biopsies and served as positive controls for comparative purposes with the DFDBA particles. Only 7 out of the 20 biopsies studied were found to have any signs of bone formation around the DFDBA particles and in these such bone formation was irregular and inconsistent around the DFDBA particles. Around the periphery of the allograft particles, osteocalcin, collagen type I, collagen type III, decorin, and biglycan all showed relatively strong staining. Osteocalcin staining was also noted within the vital bone matrix but not in the surrounding fibrous connective tissue. Decorin, biglycan, collagen type I, and collagen type III were also found within the vital bone matrix. None of these antibodies stained the DFDBA particles. The unremarkable osteogenic response of the tissues to the DFDBA particles after healing periods of up to 12 months raises questions as to the predictability of these agents in inducing new bone.

Adult↗
PubMed1996

Collagen fibril diameter distributions in ligaments and tendons of the carpal region of the horse.

Since horses bred for the racing industry are subject to rigorous training procedures there is a real need to understand how the stresses experienced by their tendons and ligaments in vivo relate to the major load-bearing elements-the collagen fibrils. Consequently, an age-related study has been made of the collagen fibril diameter distributions of nine ligaments in and around the equine carpus. This is the first stage of a larger study aimed at understanding the ultrastructural changes that occur as a result of exercise. Most of the ligaments showed a bimodal diameter distribution at maturity, and decreased diameters at old age as the fibrils break down. The scaphocapitate ligament, however, was unique in that the constituent fibrils were small, almost invariant in diameter with age, and had a unimodal distribution of sizes. The mechanical attributes of these tissues, as deduced from a theoretical analysis of the diameter distributions, are consistent with observation.

Aging↗
PubMed1995

Hard alpha-keratin IF: a structural model lacking a head-to-tail molecular overlap but having hybrid features characteristic of both epidermal keratin and vimentin IF.

In intermediate filaments (IF) both epidermal keratin and vimentin molecules have been shown to have an eight residue head-to-tail overlap between the rod domains of similarly directed molecules. In the case of the epidermal keratins this region has also been shown to have particular structural/functional significance since it represents a hot-spot for mutations in the four keratinopathies characterized to date. While there is good evidence that this head-to-tail overlap is present in IF containing Type III, IV, and V chains, as well as in the epidermal keratin IF (Ib/IIb), there are no data currently available for the hard alpha-keratin IF (Ia/IIa). Using a variety of data derived from X-ray diffraction and crosslinking studies, as well as theoretical modeling, it is now possible to demonstrate that the overlap region is not a feature of hard alpha-keratin IF. Indeed, it is shown that there is a nine residue gap between consecutive parallel molecules in the IF. An explanation for this observation is presented in terms of compensating disulfide bonds that occur both within the IF, and between the IF and the matrix in which the IF are embedded.

Amino Acid Sequence↗
PubMed1995

Hagfish biopolymer: a type I/type II homologue of epidermal keratin intermediate filaments.

In contrast to most intermediate filaments (IF) which function intracellularly or constitute epidermal appendages, the single massive (approximately 60 cm length, approximately 3 microns width) IF-rich 'thread' biopolymer synthesized by the specialized hagfish gland thread cell is released extracellularly via holocrine secretion to interact with mucins and seawater, thereby modifying the viscoelastic properties of the copious mucous exudate. Recently, using the Pacific hagfish (Eptatretus stouti, class Agnatha), a jawless scaleless marine vertebrate of ancient lineage, we determined that the deduced amino acid sequence of one thread IF chain (alpha, 66.6 kDa, native pI 7.5) contained an atypical, threonine-rich central rod domain of low identity (< 30%) with other vertebrate IF types, but that the N- and C-terminal domains exhibited several keratin-like features. From these and other unexpected characteristics, it was concluded that hagfish alpha is best categorized as a type II homologue of an epidermal keratin. We now report the deduced sequence of a second thread IF subunit (gamma, 62.7 kDa, native pI 5.3) which is co-expressed and co-assembles in vitro with alpha in a 1:1 ratio. As was found for alpha, the N- and C-terminal domains of gamma have keratin-like parameters, but the central rod has low identity to IFs of types I-V (< 31%), a cephalochordate IF (< 29%) and invertebrate IFs (< 20%) and no particular homology to type I or type II keratins. Central rod identity between gamma and alpha is also low (approximately 23%), as is typical of comparisons between different rod types but atypical of similar rod types (> 50%). The central rods of both gamma and alpha lack the 42-residue insert of helix 1B present in lamins and invertebrate IFs, have unusually high threonine contents (gamma, 10%; alpha, 13%) compared to other IF types (2-5%), contain a number of unexpected residues in consensus conserved sites, and employ a L12 segment of 21 residues rather than the 16 or 17 residues found in keratins. Theoretical analyses indicate that the hagfish molecules exist as coiled coil heterodimers (alpha/gamma) in which the chains are parallel, in axial register, and stabilized by significant numbers of ionic interactions. Fast Fourier-transform analyses revealed that the linear distribution period of approximately 9.55 for basic and acidic residues in other IF chains is not completely maintained, partly due to the high threonine content. The threonine residues occupy mainly outer sites b, c, f in the heptad substructure, possibly abetting parallel alignment of thousands of IFs within the thread, interactions with mucins at the thread periphery, and hierarchical IF chain assembly. It is suggested that the gamma and alpha chains from this most primitive extant vertebrate are type I and type II homologues of epidermal keratin chains, possibly related to early specialized keratins.

Amino Acid Sequence↗
PubMed1994

An adherens junction protein is a member of the family of lactose-binding lectins.

We previously described a pig junction protein of M(r) 37,000 found in oral epithelium but not in epidermis, limited to suprabasal cells, and colocalizing by immunofluorescence with adherens junction proteins. A 1.1-kilobase pair cDNA of the 37-kDa protein yielded an open reading frame encoding a 323-amino acid protein of 35,852 Da, and Northern analysis demonstrated a band of 1.2 kilobases in tongue RNA. Secondary structure predictions indicate that the 37% identical 16-17-kDa N- and C-terminal domains from beta-sheet-rich barrels linked by a compact proline-rich segment. The protein is 72% identical in amino acid sequence and shares symmetrical two-domain structure with L-36, a lectin of unknown function from rat intestine, indicating that the 37-kDa protein is the porcine form of L-36. Of the homologous lactose binding lectins known, two others, invertebrate lectins, share this symmetrical structure. Expression of the C-terminal domain of the pig lectin in bacteria yields a lectin which binds lactosyl-Sepharose, and binding is inhibited by lactose. The expressed protein binds a glycoprotein of 120 kDa from pig tongue epithelium on Western blots, and this is also inhibited by lactose. The findings suggest that the lectin function may be involved in the assembly of adherens junctions.

Amino Acid Sequence↗
PubMed1994

Cloning and characterization of a novel epidermal cell surface antigen (ESA).

We report here the isolation and characterization of a cDNA that encodes a novel extracellular epidermal molecule, epidermal surface antigen (ESA), which is thought to play a role in intercellular epidermal adhesion. Sequence analysis reveals that the 379 amino acid ESA has a molecular mass of about 41.7 kDa and an alpha-helix-rich secondary conformation. Much of this also has an heptad substructure, consistent with the formation of several bundles of alpha-helices in a compact globular structure. The ESA protein appears to consist of an NH2-terminal hydrophobic region with mixed alpha and beta structure followed by a more hydrophilic COOH-terminal region which is very rich in alpha-helix. The 2.5-kilobase ESA mRNA is expressed in cultured keratinocytes, melanocytes, fibroblasts, carcinoma, and melanoma cell lines. The ESA gene is conserved in all mammalian species examined and has been localized to human chromosome 17 (M17S1) in the same region as the gene for von Recklinghausen neurofibromatosis. The high level of expression of the ESA mRNA in human skin and in cultured cells derived from the epidermis, the appearance of ESA protein early in human development, and conservation of the ESA gene throughout mammalian evolution suggest that the novel ESA protein plays a vital role in epidermal structure and maintenance.

Amino Acid Sequence↗
PubMed1994

Coiled-coil stutter and link segments in keratin and other intermediate filament molecules: a computer modeling study.

Structural discontinuities have previously been identified in four regions of the coiled-coil rod domain structure present in intermediate filament (IF) protein molecules. These include a point at which a phase shift occurs in the heptad periodicity characteristic of the sequence of polar and apolar residues in alpha-helical coiled-coils, and three links that lack a heptad substructure. We have studied these regions by computer-based molecular modeling and comparative sequence analysis and conclude that the phasing discontinuity can be accommodated without significant distortion of the overall double-helical chain conformation; the L2 link has a similar conformation in all different types of IF molecules, a favorable conformation being one in which the two strands wrap tightly around each other; the L12 links vary in length between different IF types but contain important sequence similarities suggestive of a partial beta structure; the L1 links show larger variations in length, a lower degree of similarity, and probably diverse structures. Variations in the overall charges of the different links suggest that ionic interactions may play a significant role in filament assembly. The results also have general significance for other alpha-fibrous proteins in which either the characteristic heptad phasing undergoes a discontinuity or where a short non-coiled-coil sequence occurs within a coiled-coil rod domain structure.

Amino Acid Sequence↗
PubMed1994

Structure and function of desmosomal transmembrane core and plaque molecules.

Desmosomes are intercellular junctions that function in cell-cell adhesion and attachment of intermediate filaments (IF) to the cell surface. Desmogleins and desmocollins are the major components of the transmembrane adhesion complex, whereas desmoplakins (DPs) are the most prominent components of the cytoplasmic plaque. Based on sequence similarity, desmogleins and desmocollins are related to the calcium-dependent homophilic adhesion molecules known as cadherins. Like the classical cadherins, the desmosomal cadherins contain four homologous extracellular domains bearing putative calcium-binding sites, a single transmembrane spanning domain, and a C-terminal cytoplasmic tail. Molecules in the desmoglein subclass contain a unique C-terminal extension within which is found a repeating motif that is predicted to form two beta-strands and two turns. Stable cell lines expressing desmoglein 1 have been generated from normally non-adherent L cell fibroblasts, to study the contribution of this cadherin to desmosomal adhesion. The predicted sequence of desmoplakin (DP) I suggests it will form homodimers comprising a central alpha-helical coiled-coil rod and two globular end domains. The C-terminus contains three regions with significant homology, each of which is made up of a 38-residue motif also found in two other molecules involved in organization of IF, bullous pemphigoid antigen and plectin. Ectopically expressed polypeptides including the C-terminus of DP I specifically align with keratin and vimentin IF in cultured cells, whereas those lacking this domain do not align with IF. The last 68 amino acids of DP are required for alignment along keratin but not vimentin IF, and residues 48-68 from the C-terminal end are critical for this interaction. These results suggest that the C-terminus of DP plays a role in the attachment of IF to the desmosome and that a specific site is necessary for interaction with keratin IF. A sequence at the most N-terminal end of DP appears to be required for efficient incorporation into the desmosomal plaque. Interestingly, this region has not been reported to be present in the homologous bullous pemphigoid antigen or plectin molecules and may represent a desmosomal targeting sequence.

Animals↗
PubMed1994

NuMA/centrophilin: sequence analysis of the coiled-coil rod domain.

Nuclear mitotic apparatus protein (NuMA), also known as centrophilin, has been shown in previous work to contain a centrally located sequence of length 1485 residues that has both a heptad substructure and a high propensity for alpha-helix formation. Further analysis of this sequence here has revealed that NuMA will form a two-stranded coiled-coil structure with multiple (18) points at which the conformation is interrupted either by proline-containing segments or by discontinuities in the phasing of the heptad substructure. It has also been shown that the two chains will be parallel (rather than antiparallel), that they will lie in axial register, and that this arrangement will be stabilized by a large number of interchain ionic interactions. Interestingly the coiled-coil rod domain is also shown to lack any significant long-range periodicity in the linear distribution of either its acidic or its basic residues. Hence there is no direct evidence from the sequence data that NuMA molecules will aggregate to form closely packed filaments within nuclear space.

Amino Acid Sequence↗
PubMed1994

Structural features of keratin intermediate filaments.

The first step in the assembly of a keratin intermediate filament (KIF) is the formation of a type I/type II heterodimer molecule in which two chains become aligned in parallel and close axial registration to form a flexible segmented alpha-helical coiled-coil rope 46 nm long. The segments of coiled-coil are interspersed by sequences that introduce irregularities of unknown structure. Here we have modeled two of these, the link L2 and the heptad discontinuity located near the middle of segment 2B. In a model for L2, the orientation of the coiled-coil structure is turned through about 180 degrees over the eight residue stretch constituting this link segment. In contrast, the heptad discontinuity in segment 2B would seem to result in only minimal distortion of the coiled-coil rope, contrary to previous expectations. Little is known about how the neighboring molecules are aligned and packed within the assembled KIF. Crosslinking experiments with KIF have determined that two neighboring molecules are aligned anti-parallel and axially in three ways, and predict that similarly-directed molecules could be overlapped by about 1 nm. The two-dimensional surface lattice resulting from these data predicts an axial periodicity of 22.6 nm, which in fact is visible by electron microscopy of shadowed KIF. Interestingly, most of the amino acid substitutions resulting from mutations in the keratin genes found in genodermatoses are clustered in this molecular overlap region. Although we do not yet know how the rows of antiparallel molecules fold in three dimensions to form an intact KIF, certain of the observed crosslinks could also occur between nearest neighbor parallel molecules across a four-molecule strand; that is, KIF may be built from bundles or protofibrils. These insights on molecular structure and molecular packing provide new constraints on models for KIF structure.

Amino Acid Sequence↗
PubMed1994

An unusual intermediate filament subunit from the cytoskeletal biopolymer released extracellularly into seawater by the primitive hagfish (Eptatretus stouti).

Each slime gland thread cell from the primitive Pacific hagfish (Eptatretus stouti) contains a massive, conical, intermediate filament (IF)-rich biopolymer ('thread,' approximately 60 cm length, approximately 3 microns width). In view of the unusual ultrastructure of the thread, its extracellular role in modulation of the viscoelastic properties of mucus, and the ancient lineage of this primitive vertebrate, we report the nucleotide and deduced amino acid sequences of one major thread IF subunit, alpha (pI 7.5), which is coexpressed with a second polypeptide, gamma (pI 5.3). These two polypeptides coassemble in vitro into approximately 10 nm filaments. The alpha-thread chain, a 66.6 kDa polypeptide, has an unusual central rod domain containing 318 residues flanked by N- and C-terminal domains of 192 and 133 residues, respectively. Each peripheral region exhibits some epidermal keratin-like features including peptide repeats and a high total content of glycine and serine residues. The terminal domains, however, lack the H1 and H2 subdomains characteristic of known keratins. Moreover, when the central rod is aligned either in relation to established homology profiles (J. F. Conway and D. A. D. Parry (1988) Int. J. Biol. Macromol. 10, 79-98) of other IF subunits (types I-V, nestin, non-neuronal invertebrate), or by computer-based homology searches of the GenBank/EMBL Data Bank, a low identity (< 30%) is evident, with no preferred identity to keratins or other known IF types. Although the central rod of 318 residues consists of the canonical apolar heptad repeats interspersed with three linker regions, a discontinuity in phasing of the heptad substructure in rod 2B, and conserved sequences at either end of the rod domain, other collective characteristics are atypical: overall high threonine content (13.2% vs 2.3-5.4% for other IFs), high threonine content in rod 1B (18.8% vs 1-6%), five Thr-Thr repeats in coiled coil segments, L12 of length greater than in keratins, substitution of phenylalanine for a highly conserved glutamate in the sixth position of L2, and a glycine-proline sequence in segment 2B. Possibly as a result of the high threonine content, the percentage of both acidic and basic residues in most helical subdomains is reduced relative to type I and II chains. Fast Fourier transform analyses show that only the acidic residues in segment 1B and basic residues in segment 2 have near typical IF periods.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Fatal methemoglobinemia due to inhalation of isobutyl nitrite.

Isobutyl nitrite is a popular recreational drug among both homosexuals and heterosexuals as it is alleged to enhance sexual pleasure and prolong orgasm. In contrast to the ingestion of this volatile nitrite, inhalation is associated only rarely with serious sequelae, though symptomatic methemoglobinemia may occur. The case reported is the first documented fatality from inhalation of isobutyl nitrite. The widespread use of isobutyl nitrite is a cause for concern and physicians should be aware of the potentially fatal consequence of abuse, particularly in those with ischemic heart disease, and its management.

Administration, Inhalation↗
PubMed1993

Diversity of intermediate filament structure. Evidence that the alignment of coiled-coil molecules in vimentin is different from that in keratin intermediate filaments.

Although vimentin intermediate filaments (IF) are morphologically similar to all other IF types, cells have evolved different ways of manipulating vimentin and keratin IF. The structural basis for such differences is unknown. We have explored this by use of cross-linking experiments on vimentin oligomers, polymers, and intact IF to determine the axial length of vimentin molecules and the degrees to which neighboring molecules are aligned in IF. Our data reveal that the homodimer vimentin molecule (43.9 nm) is clearly shorter than a keratin heterodimer molecule (46.2 nm). Vimentin assemblies contain three modes of antiparallel molecular alignments: A11 and A22 in two-molecule or larger oligomeric assemblies, in which the two molecules are staggered so as to bring their 1B and 2B rod domain segments, respectively, into register; and A12 in higher order molecular assemblies in which the two neighboring molecules are largely overlapped. Since the repeat axial length of the vimentin assemblies (42.6 nm) is less than the molecular length, this means there is an overlap (designated as alignment ACN) of about 1 nm (5-10 residues) between the end of the 2B and beginning of the 1A rod domain segments of similarly directed molecules in the IF. Interestingly, these four modes of nearest neighbor molecular alignments also occur in keratin IF. However, the degree of stagger of alignments in the A11 and A22 modes is different (staggers of -19.5 for vimentin versus -16.6 nm for keratin, and 23.3 and 28.6 nm, respectively). Two-dimensional surface lattice maps of the two IF types are very similar, except for differences in molecule alignments and different axial repeats of 21.4 nm in vimentin and 22.6 nm in keratin IF. Although vimentin-keratin hybrid molecules can be induced to form in vitro, they do not assemble into higher order structures. The data suggest that vimentin and keratin are incapable of assembly into IF in vitro or in vivo simply because their molecules are of different axial lengths and because the exact axial alignments of neighboring molecules are different.

Amino Acid Sequence↗
PubMed1993

Conservation of the structure of keratin intermediate filaments: molecular mechanism by which different keratin molecules integrate into preexisting keratin intermediate filaments during differentiation.

During development and differentiation, the intermediate filament component of the cytoskeleton of many cells and tissues is rebuilt by a dynamic exchange process in which one set of protein chains is replaced by another, without recourse to creation of a new network. One major example is the replacement of keratin 5/keratin 14 (K5/K14) keratin intermediate filaments (KIFs) by K1/K10 KIFs during terminal differentiation in the epidermis. The present work was undertaken to explore how this may occur. We have induced lysine-lysine cross-links with disulfosuccinimidyl tartrate in K5/K14 KIFs in order to determine the axial dimensions and relative axial alignments of the K5/K14 molecules. Many of the cross-links induced in subfilamentous oligomers containing one, two, or three molecules were also found in the intact KIF, indicating that the body of data thus generated provides physiologically relevant information on the structural organization in the KIF. A least-squares analysis using as data the positions of lysine residues involved in 23 induced cross-links has allowed the axial alignments of the various coiled-coil segments in the rod domain to be determined. Three modes of antiparallel alignment of two neighboring molecules were found: A11 (staggered by -16.7 nm), A22 (staggered by 28.8 nm), and A12 (almost in register; staggered by only 0.3 nm). Since the axial repeat length is about 1 nm less than the molecular length, the data require a fourth mode of molecule alignment, termed ACN, in which similarly directed molecules are overlapped by the equivalent of about 5-10 residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

The structure of human trichohyalin. Potential multiple roles as a functional EF-hand-like calcium-binding protein, a cornified cell envelope precursor, and an intermediate filament-associated (cross-linking) protein.

Trichohyalin is an intermediate filament-associated protein that associates in regular arrays with keratin intermediate filaments (KIF) of the inner root sheath cells of the hair follicle and the granular layer of the epidermis and is a known substrate of transglutaminases. We have determined the full-length sequence of human trichohyalin by use of RNA-mediated anchored polymerase chain reaction methods and from a genomic clone and analyzed its potential secondary structure. We show here that trichohyalin may have at least three important functions in these cells. The protein of 248 kDa is unusual in that it contains one of the highest contents of charged residues of any protein. Of several defined domains, domains 2-4, 6, and 8 are almost entirely alpha-helical, configured as a series of peptide repeats of varying regularity, and are thought to form a single-stranded alpha-helical rod stabilized by ionic interactions between successive turns of the alpha-helix. Domain 6 is the most regular and may bind KIF directly by ionic interactions. Domains 5 and 7 are less well organized and may introduce folds in the molecule. Thus, human trichohyalin is predicted to be an elongated flexible rod at least 215 nm long and to function as a KIF-associated protein by cross-linking the filaments in loose networks. In addition, trichohyalin is similar to, but several times longer than, involucrin, a known cell envelope constituent, so that together, involucrin and trichohyalin may serve as scaffold proteins in the organization of the cell envelope of these cells or even anchor the cell envelope to the KIF network. Finally, trichohyalin possesses a pair of functional calcium-binding domains of the EF-hand type at its amino terminus that may be involved in its calcium-dependent postsynthetic processing during terminal differentiation.

Amino Acid Sequence↗
PubMed1993

Ionic interactions in the coiled-coil domain of laminin determine the specificity of chain assembly.

Laminins are a family of large (800 to 900 kDa) multidomain glycoproteins specifically found in basement membranes. They consist of one heavy A chain and two light chains B1 and B2, and several tissue-specific laminin isoforms exist. Chain assembly is mediated through the formation of a rod-like triple-stranded alpha-helical coiled-coil domain about 75 nm long. The interacting edges of the chains are mostly formed by hydrophobic residues in positions a and d of an (abcdefg)n heptad sequence repeat and by a distinct pattern of charged residues in positions e and g. Here, we have analyzed the sequences of known laminin chains in an effort to relate them to interaction potential. Initially, those sequences localized in the long arm were arranged in an optimum heptad-repeating scheme. The interacting edges between chains were then analyzed for interchain hydrophobic and ionic interactions. The short heptad blocks were allowed to shift axially with respect to each other to maximize the number of interactions. The number of hydrophobic interactions was very high and similar for all chain combinations, but especially so for homodimers. As these were not observed experimentally, it seems that hydrophobic interactions probably represent only a prerequisite for coiled-coil formation. The number of ionic interactions, however, directly resembles the interaction potential observed in in vitro experiments. In particular, the number of interchain ionic interactions is high for parallel heterodimer configurations of A and B chains, but low for homodimer arrangements. When the laminin isoform chains, rat s-laminin (B1s) and human merosin (Am), are included in the analysis, they show rather low numbers of mutual interactions but high ionic interaction potentials between them and distinct mouse laminin chains are predicted. For mouse laminin the analysis was extended to a full three-stranded coiled-coil structure. The highest number of interchain ionic interactions occurs for an anti-clockwise chain arrangement of A-->B1-->B2 when viewed from the N terminus. None of the laminin chains appears to be designed for the formation of homodimers, although such conformations are frequently found in other alpha-fibrous proteins.

Amino Acid Sequence↗
PubMed1993

Keratin intermediate filament structure. Crosslinking studies yield quantitative information on molecular dimensions and mechanism of assembly.

One of the major obstacles to solving the full three-dimensional structure of keratin intermediate filaments (KIF) is the determination of the exact mode(s) of alignment of nearest-neighbor molecules; this in turn requires precise information of the lengths of the non-alpha-helical linker segments within the coiled-coil alpha-helical heterodimer molecule. In this study, we have induced lysine-lysine and cysteine-cysteine crosslinks between keratin intermediate filament molecules in small assembly-competent oligomers, isolated them and then characterized the natures and locations of the crosslinks. Of more than 100 found, 21 quantitatively major crosslinks were used to obtain the relative axial alignments of rod domain segments by least-squares fitting methods. Three dominant modes of alignment were found. In each case the molecules are antiparallel with the first involving molecules in approximate register (stagger = -0.2 nm), the second involving molecules staggered so as to bring the 1B segments into approximate alignment (stagger = -16.1 nm), and the third involving molecules staggered so as to bring the 2B segments into approximate alignment (stagger = 28.2 nm). In addition, the data enable quantitative estimates to be made for the first time of the lengths of the non-coiled-coil segments (L1 = 2.5 nm, L12 = 1.6 nm, L2 = 0.8 nm), and the total length of the rod domain (46.0 nm). Alignment of molecules according to these parameters permits construction of a two-dimensional surface lattice which displays a 1.6 nm (10 or 11 residue) overlap between similarly directed molecules. Together, the data predict six important overlapping sequence regions that recur about 16 times per 46 nm of filament length. Interestingly, synthetic peptides corresponding to these sequences, singly or in combination, significantly interfere with keratin filament structural integrity. These results thus represent the most significant set of structural constraints for KIF yet available and provide insights into how disease-causing mutations disrupt filaments and their organization in cells.

Amino Acid Sequence↗