Matrilins: structure, expression and function.
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Animals↗
Biomedical subjects
N Smyth
Publications and source records attributed to N Smyth.
Loss of annexin A7 leads to alterations in frequency-induced shortening of isolated murine cardiomyocytes.
Annexin A7 has been proposed to function in the fusion of vesicles, acting as a Ca(2+) channel and as Ca(2+)-activated GTPase, thus inducing Ca(2+)/GTP-dependent secretory events. To understand the function of annexin A7, we have performed targeted disruption of the Anxa7 gene in mice. Matings between heterozygous mice produced offspring showing a normal Mendelian pattern of inheritance, indicating that the loss of annexin A7 did not interfere with viability in utero. Mice lacking annexin A7 showed no obvious phenotype and were fertile. To assay for exocytosis, insulin secretion from isolated islets of Langerhans was examined. Ca(2+)-induced and cyclic AMP-mediated potentiation of insulin secretion was unchanged in the absence of annexin A7, suggesting that it is not directly implicated in vesicle fusion. Ca(2+) regulation studied in isolated cardiomyocytes, showed that while cells from early embryos displayed intact Ca(2+) homeostasis and expressed all of the components required for excitation-contraction coupling, cardiomyocytes from adult Anxa7(-/-) mice exhibited an altered cell shortening-frequency relationship when stimulated with high frequencies. This suggests a function for annexin A7 in electromechanical coupling, probably through Ca(2+) homoeostasis.
Animals↗
Thrombospondin-4 binds specifically to both collagenous and non-collagenous extracellular matrix proteins via its C-terminal domains.
Full-length and truncated forms of rat thrombospondin-4 (TSP-4) were expressed recombinantly in a mammalian cell line and purified to homogeneity. Biochemical analysis revealed a limited proteolytic processing, which detaches the N-terminal heparin-binding domain from the rest of the molecule and confirmed the importance of the heptad-repeat domain for pentamerization. In electron microscopy the uncleaved TSP-4 was seen as a large central particle to which five smaller globules are attached by elongated linker regions. Binding of TSP-4 to collagens and to non-collagenous proteins could be detected in enzyme-linked immunosorbent assay-style ligand binding assays, by surface plasmon resonance spectroscopy, and in rotary shadowing electron microscopy. Although the binding of TSP-4 to solid-phase collagens was enhanced by Zn(2+), that to non-collagenous proteins was not. The interactions of TSP-4 with both classes of proteins are mediated by C-terminal domains of the TSP-4 subunits but do not require an oligomeric structure. Major binding sites for TSP-4 are located in or close to the N- and C-terminal telopeptides in collagen I, but additional sites are detected in more central regions of the molecule.
Amino Acid Sequence↗
Laminins: structure and genetic regulation.
The laminins form a large family of modular proteins found in basement membranes, but also elsewhere. They function as structural components and are essential for morphogenesis, but in addition interact with cell surface receptors such as integrins and alpha-dystroglycan. By virtue of their receptor interactions, they initiate intracellular signalling events that regulate cellular organization and differentiation. The many interactions of laminins are mediated by binding sites, often contributed by single domains, which may differ between different forms of laminin. In the present article, we describe how the diversity of laminins and the genetic regulation of the expression of different laminin forms lead to the formation of extracellular matrices with variable laminin composition and thereby different biological properties.
Alternative Splicing↗
The LIM-only protein DRAL/FHL2 binds to the cytoplasmic domain of several alpha and beta integrin chains and is recruited to adhesion complexes.
LIM proteins contain one or more double zinc finger structures (LIM domains) mediating specific contacts between proteins that participate in the formation of multiprotein complexes. We report that the LIM-only protein DRAL/FHL2, with four and a half LIM domains, can associate with alpha(3A), alpha(3B), alpha(7A), and several beta integrin subunits as shown in yeast two-hybrid assays as well as after overexpression in human cells. The amino acid sequence immediately following the conserved membrane-proximal region in the integrin alpha subunits or the C-terminal region with the conserved NXXY motif of the integrin beta subunits are critical for binding DRAL/FHL2. Furthermore, the DRAL/FHL2 associates with itself and with other molecules that bind to the cytoplasmic domain of integrin alpha subunits. Deletion analysis of DRAL/FHL2 revealed that particular LIM domains or LIM domain combinations bind the different proteins. These results, together with the fact that full-length DRAL/FHL2 is found in cell adhesion complexes, suggest that it is an adaptor/docking protein involved in integrin signaling pathways.
3T3 Cells↗
Expression of matrilin-1, -2 and -3 in developing mouse limbs and heart.
The expression of matrilin-1, -2 and -3 was studied in the heart and limb during mouse development. Matrilin-1 is transiently expressed in the heart between days 9.5 and 14.5 p.c. Matrilin-2 expression was detected in the heart from day 10.5 p.c. onwards. In the developing limb bud, both matrilin-1 and -3 were observed first at day 12.5 p.c. Throughout development matrilin-3 expression was strictly limited to cartilage, while matrilin-1 was also found in some other forms of connective tissue. Matrilin-2, albeit present around hypertrophic chondrocytes in the growth plate, was mainly expressed in non-skeletal structures. The complementary, but in part overlapping, expression of matrilins indicates the possibility for both redundant and unique functions among the members of this novel family of extracellular matrix proteins.
Animals↗
The absence of nidogen 1 does not affect murine basement membrane formation.
Nidogen 1 is a highly conserved protein in mammals, Drosophila melanogaster, Caenorhabditis elegans, and ascidians and is found in all basement membranes. It has been proposed that nidogen 1 connects the laminin and collagen IV networks, so stabilizing the basement membrane, and integrates other proteins, including perlecan, into the basement membrane. To define the role of nidogen 1 in basement membranes in vivo, we produced a null mutation of the NID-1 gene in embryonic stem cells and used these to derive mouse lines. Homozygous animals produce neither nidogen 1 mRNA nor protein. Surprisingly, they show no overt abnormalities and are fertile, their basement membrane structures appearing normal. Nidogen 2 staining is increased in certain basement membranes, where it is normally only found in scant amounts. This occurs by either redistribution from other extracellular matrices or unmasking of nidogen 2 epitopes, as its production does not appear to be upregulated. The results show that nidogen 1 is not required for basement membrane formation or maintenance.
Animals↗
Eukaryotic expression and purification of recombinant extracellular matrix proteins carrying the Strep II tag.
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Animals↗
Absence of basement membranes after targeting the LAMC1 gene results in embryonic lethality due to failure of endoderm differentiation.
The LAMC1 gene coding for the laminin gamma1 subunit was targeted by homologous recombination in mouse embryonic stem cells. Mice heterozygous for the mutation had a normal phenotype and were fertile, whereas homozygous mutant embryos did not survive beyond day 5.5 post coitum. These embryos lacked basement membranes and although the blastocysts had expanded, primitive endoderm cells remained in the inner cell mass, and the parietal yolk sac did not develop. Cultured embryonic stem cells appeared normal after targeting both LAMC1 genes, but the embryoid bodies derived from them also lacked basement membranes, having disorganized extracellular deposits of the basement membrane proteins collagen IV and perlecan, and the cells failed to differentiate into stable myotubes. Secretion of the linking protein nidogen and a truncated laminin alpha1 subunit did occur, but these were not deposited in the extracellular matrix. These results show that the laminin gamma1 subunit is necessary for laminin assembly and that laminin is in turn essential for the organization of other basement membrane components in vivo and in vitro. Surprisingly, basement membranes are not necessary for the formation of the first epithelium to develop during embryogenesis, but first become required for extra embryonic endoderm differentiation.
Animals↗
The matrilins: a growing family of A-domain-containing proteins.
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Animals↗
Recombinant human tissue transglutaminase ELISA for the diagnosis of gluten-sensitive enteropathy.
BACKGROUND: Tissue transglutaminase (TGc) has recently been identified as the major, if not the sole, autoantigen of gluten-sensitive enteropathy (GSE). We developed and validated an ELISA based on the human recombinant antigen and compared it to existing serological tests for GSE [guinea pig TGc ELISA and endomysium antibody (EMA) test]. METHODS: Human TGc was expressed in the human embryonic kidney cell line 293-EBNA as a C-terminal fusion protein with the eight-amino acid Strep-tag II allowing one-step purification via streptavidin affinity chromatography. We carried out ELISA assays for IgA antibodies against TGc using calcium-activated human and guinea pig TGc. The sera were also tested on monkey esophagus sections by indirect immunofluorescence for IgA EMA. We examined 71 serum samples from patients with GSE (38 with celiac disease, 33 with dermatitis herpetiformis), including 16 on therapy, and 53 controls. RESULTS: The human TGc could be expressed and purified as an active enzyme giving a single band on a Coomassie-stained gel. The mean intra- and interassay CVs for the human TGc ELISA were 3.2% and 9.2%, respectively. The area under the ROC curve was 0.999. The specificity and sensitivity were 98.1% (95% confidence interval, 95.7-100%) and 98.2% (95.9-100%), respectively. CONCLUSIONS: The human TGc ELISA was somewhat superior to the guinea pig TGc ELISA, and was as specific and sensitive as the EMA test. The human TGc-based ELISA is the method of choice for easy and noninvasive screening and diagnosis of GSE.
Adolescent↗
The targeted deletion of the LAMC1 gene.
Laminin is required for the production of a basement membrane in both the early embryo and in the embryoid body. The absence of the gamma 1 chain leads to different processing of the other subunits in the trimer and prevents the production of a polymerizing laminin molecule. Collagen IV and perlecan are not able to compensate for the loss of the laminin molecule, and nidogen, which would normally bind to the gamma 1 chain, is almost completely lost from the embryoid body. In the embryo the failure of the basement membrane results in embryonic lethality at embryonic day 5.5, a time when the primitive endoderm is differentiating to visceral and parietal endoderm, and the latter is migrating out of the inner cell mass over the trophectoderm. It is likely that failure in one or both of these events leads to the death of the embryo. The absence of the trophectodermal basement membrane normally present in the blastocyst appears not to be necessary for the formation of the blastocystic cavity, which depends on the formation of a polarized epithelium. Hence the first epithelium produced in the developing embryo does so independently of a basement membrane.
Animals↗
The role of laminins in basement membrane function.
Laminins are a family of multifunctional macromolecules, ubiquitous in basement membranes, and represent the most abundant structural noncollagenous glycoproteins of these highly specialised extracellular matrices. Their discovery started with the difficult task of isolating molecules produced by cultivated cells or extracted from tissues. The development of molecular biology techniques has facilitated and accelerated the identification and the characterisation of new laminin variants making it feasible to identify full-length polypeptides which have not been purified. Further, genetically engineered laminin fragments can be generated for studies of their structure-function relationship, permitting the demonstration that laminins are involved in multiple interactions with themselves, with other components of the basal lamina, and with cells. It endows laminins with a central role in the formation, the architecture, and the stability of basement membranes. In addition, laminins may both separate and connect different tissues, i.e. the parenchymal and the interstitial connective tissues. Laminins also provide adjacent cells with a mechanical scaffold and biological information either directly by interacting with cell surface components, or indirectly by trapping growth factors. In doing so they trigger and control cellular functions. Recently, the structural and biological diversity of the laminins has started to be elucidated by gene targeting and by the identification of laminin defects in acquired or inherited human diseases. The consequent phenotypes highlight the pivotal role of laminins in determining heterogeneity in basement membrane functions.
Animals↗
Laminin heterogeneity around Schlemm's canal in normal humans and glaucoma patients.
BACKGROUND: The laminins play an important role in cell attachment to the extracellular matrix. This study was undertaken in order to investigate and to compare the presence of several laminin chains and isoforms in normal and glaucomatous trabecular meshwork. METHODS: Fresh-frozen trabeculectomy specimens from glaucoma patients and trabecular meshwork from human donor eyes were included for immunohistochemistry. We used antibodies against laminin 1 (EHS-laminin/alpha1beta1gamma1), laminin 2 (merosin/alpha2,beta1gamma1), the subunits of alpha2 (M laminin), beta1 B1laminin), gamma1 (beta2laminin) and beta2 (s laminin) and gamma2 and the associated glycoprotein nidogen. RESULTS: For laminin 1, laminin 2, the subunits alpha2;, beta1, beta2, gamma1 and nidogen, the immunoreactivity of the juxtacanalicular zone underneath the inner wall of Schlemm's canal was always stronger than of the uveal and corneoscleral trabecular meshwork. Staining for laminin chain gamma2 could not be demonstrated in the outflow structures. Pattern and intensity of labelling for the laminins were not different between normal and glaucomatous eyes. DISCUSSION: Heterogeneity of the laminins suggests structural complexity of the juxtacanalicular region. However, the presence of laminin around Schlemm's canal seems to be unaffected by glaucoma and by age. This might indicate maintenance of basement membrane stability in the trabecular meshwork for life.
Adult↗
[Variability of laminin isoforms in the limbus region of the eye].
BACKGROUND: Cell adhesion in the limbal region is of outstanding importance for the regeneration of the corneal epithelium and for repair mechanisms after antiglaucomatous fistulating surgery. In the basement membranes cell adhesion is largely modified by the extracellular matrix protein laminin. The aim of our study was to establish the immunohistochemical pattern of the different laminin-isoforms and subunits in the basal membrane of the limbal conjunctiva and episcleral vessels. MATERIAL AND METHOD: For immunohistochemistry five normal human donor eyes were included; we used antibodies against the laminin heterotrimers 1 and 2, against the laminin subunits alpha 2, beta 1, beta 2, gamma 1, gamma 2 and against the laminin-associated protein nidogen. RESULTS: The basement membrane of the limbal conjunctiva reveals immunoreactivity against all used antibodies. The subconjunctival and episcleral vessels showed no staining for the laminin subunit gamma 2, but for all other used antibodies. CONCLUSION: The basement membrane of the limbal and conjunctival epithelium as well as the basement membrane of subconjunctival and episcleral vessels express a broad spectrum of laminin variants. This diversity emphasizes functional specialization of the limbal region, although the exact importance of the laminin variants is still unknown.
Adult↗
Regulated expression of a novel laminin beta subunit during the development of the chick embryo.
In order to define specific laminin variants implicated in organogenesis, we have undertaken a systematic search to detect and characterize novel laminin subunits, the expression of which is both developmentally regulated and tissue-specific. cDNA prepared from embryonic chick tissues was amplified by the polymerase chain reaction (PCR) with degenerate primers based on conserved sequences in domains V and VI of members of the laminin beta subunit family. Restriction mapping, cloning and sequencing of PCR products demonstrated a novel cDNA, the derived protein sequence of which displayed greatest homology with laminin beta subunits. However, the degree of amino acid divergence, comparison of sequence motifs and pattern of expression indicates that the cloned cDNA does not code for the avian orthologue of a previously characterized laminin beta subunit. Northern blot analysis showed that the expression of the 6-kb mRNA coding for the novel subunit was restricted to the skin. The mRNA was not detectable before day 5 of chick embryonic development, after which in situ hybridization showed expression only in surface ectoderm cells and subsequently in the epidermis. The developmentally regulated ectodermal expression of the novel beta subunit, prior to condensation of mesenchymal cells to form the dermis, is consistent with a specific role for this laminin isoform in the development and maintenance of the skin.
Amino Acid Sequence↗
Management of aortic arch aneurysm using profound hypothermia and circulatory arrest.
The cases of 9 patients with aneurysms involving the aortic arch, repaired under profound hypothermia (average, 15.5 degrees C) and circulatory arrest, are presented. Five patients underwent elective operation and 4, emergency operation. Arch resection and graft replacement were done in 7 patients. Two patients with infected pseudoaneurysms of the aortic arch received patch grafts. There were 2 deaths (22%) from coagulopathy and decerebration. Seven patients are alive and well 18 to 45 months following repair. The combination of profound hypothermia and circulatory arrest appears to be a promising solution to a difficult problem.
Adult↗
Those lollies tasted nice.
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Accident Prevention↗