Structural analysis and proteolytic processing of recombinant G domain of mouse laminin α2 chain

AbstractFour individual LG modules from the C-terminus of the laminin α2 chain (LG1, LG2, LG4 and LG5) and combinations of these modules were prepared as recombinant products from transfected mammalian cells. This demonstrated that LG modules represent autonomously folding protein domains. Successful production depended on proper alignment of module borders and required a sequence correction at the C-terminus which added an extra cysteine. The LG modules were glycosylated and shown by electron microscopy to have a globular shape, indicating proper folding. Evidence is provided for the splicing of a 12 bp exon in LG2, although this did not impair folding. Proteolytic cleavage at the C-terminus of a basic sequence was observed close to the N-terminus of LG3. A similar processing also occurs in tissue-derived laminin-2 and -4 which contain the α2 chain

The N-terminal globular domain of the laminin α1 chain binds to α1β1 and α2β1 integrins and to the heparan sulfate-containing domains of perlecan

AbstractThe N-terminal domains VI plus V (62 kDa) and V alone (43 kDa) of the laminin α1 chain were obtained as recombinant products and shown to be folded into a native form by electron microscopy and immunological assays. Domain VI alone, which corresponds to an LN module, did not represent an autonomously folding unit in mammalian cells, however. Fragment α1VI/V, but not fragment α1V, bound to purified α1β1 and α2β1 integrins, to heparin, and to heparan sulfate-substituted domains I and V of perlecan. This localized the binding activities to the LN module, which contains two basic sequences suitable for heparin interactions

Cell adhesion and integrin binding to recombinant human fibrillin-1

AbstractFibrillin-1 is a major constituent of tissue microfibrils that occur in most connective tissues, either in close association with or independent of elastin. To test possible cell-adhesive functions of this protein, we used recombinant human fibrillin-1 polypeptides produced in a mammalian expression system in cell attachment and solid-phase integrin binding assays. Fibrillin-1 polypeptides containing the single RGD sequence located in the fourth 8-cysteine domain, mediated distinct cell adhesion of a variety of cell lines and bound to purified integrin αVβ3. Integrins αIIbβ3, α5β1, α2β1 and α1β1 did not interact with any of the recombinant fibrillin-1 peptides. Our results indicate a novel role for fibrillin-1 in cellular interactions mediated via an RGD motif that is appropriately exposed for recognition by integrin αVβ3

The time-dependent rearrangement of the epithelial basement membrane in human skin wounds

In 62 human skin wounds (surgical wounds, stab wounds and lacerations after surgical treatment) we analyzed the immunohistochemical localization of collagen IV in the epithelial basement membrane. In 27 of these wounds the distribution of collagen VII, which represents a specific component of the basement membrane of stratified epithelia, was also analyzed. We were able to demonstrate a virtually identical co-distribution of both collagen IV and VII in the wound area with no significant time-dependent differences in the appearance of both collagen types. Fragments of the epithelial basement membrane could be detected in the wound area from as early as 4 days after wounding and after 8 days a complete restitution of the epithelial basement membrane was observed. In all cases with a wound age of more than 21 days the basement membrane was completely reformed over the former lesional area. The period between 8 and 21 days after wounding was characterized by a wide variability ranging from complete restitution to deposition of basement membrane fragments or total lack of the epidermal basement membrane

Isolation, characterization, and substrate properties of the external limiting membrane from the avian embryonic optic tectum

The external limiting membrane of the avian embryonic optic tectum is isolated by mechanically separating the neuronal mesencephalon from the overlying mesenchymal tissue. The preparation consists of a basal lamina which is covered on its neural side by endfeet of neuroepithelial cells and has attached to it on its meningeal side a collageneous stroma, containing blood vessels. The external limiting membrane can be flat-mounted on a piece of nitrocellulose filter as mechanical support. It covers an area between 0.3 and 1 the cm2, depending on the age of me donor embryo. The endfeet can be removed together with all cellular components of the meninges by treatment with 2% Triton-X-100 or with distilled water. The basal lamina itself is approximately 80 nm thick and consists of two laminae rarae and a central lamina densa. Immunohistochemical staining reveals that the basal lamina in the embryo, after isolation and after detergent extraction of the isolated preparation, contains type IV collagen, nidogen, laminin, and low density heparan sulfate proteoglycan as do other basement membranes. Antibodies against the neural cell adhesion molecule (N-CAM), chondroitin sulfate proteoglycan, and fibronectin fail to stain the external limiting membrane, but these proteins were clearly identified in the blood vessel-containing meninges or in the optic tectum. The flat-mounted external limiting membrane preparation was used as substrate to culture several different neural tissues of central and peripheral origin. Explants of neural crest cells, dorsal root ganglia, and sympathetic ganglia can be cultured on the external limiting membrane. All explants grow well on the basal lamina preparations whether the endfeet are attached or detergent-extracted prior to explantation; however, neurite outgrowth from sympathetic ganglia is reduced in the presence of the endfeet. Although the endfoot-lined external limiting membrane represents at least part of the immediate environment encountered by retinal axons as they invade the optic tectum and despite its excellent properties as a substrate for retinal axons in vitro, cues guiding the orientation of axons were not detected in the flat-mounted preparation

Biochemical Composition of the Epidermal-dermal Junction and Other Basement Membrane

The epidermo-dermal junction ultrastructurally appears to be high organized, presenting 4 layers: (1) the basal cell plasma membrane, a trilaminar layer with special attachment structures (hemidesmosomes); (2) lamina lucida (rara) an electron-lucent zone with 'anchoring' filaments (vide infra); (3) basal lamina (densa) electron-dense and relatively thick (30-50 nm) and (4) the subbasal fibrous area, composed of 3 different fibrils: (a) 'anchoring fibrils' meshing with the lamina densa upwards and forming and interlocking (a) meshwork with the adjacent anchoring fibrils downwards; (b) dermal microfibril bundles starting from the basal lamina and passing deep in the dermis. The dermal part of these, are in connection with the elastic fibers and ultrastructurally they are identical and; (c) collagen fibers, randomly oriented without connection with the basal lamina. The biochemical composition comprises collagenous and noncollagenous substances: collagens can be divided into 3 groups designated as type IV collagen, type V(AB collagen) and 7S collagen. The non-collagenous materials as yet identified are: bullous pemphigoid antigen (reactive with the bullous pemphigoid antibodies), laminin, heparan-sulfate containing proteoglycan and amyloid P-component (a normal plasma constituent). Functional aspects of basement membrane components: (1) support of a caffolding function assumed by the collagens; (2) attachment of cells of different embryonic origin (fibronectin, PB antigen and laminin); (3)regulation of the permeability (heparan-sulfate proteoglycan and other glycosaminoglycans); and (4) a role in development and morphogenesis (laminin) and type IV collagen). This paper must be read in extenso by those interested in the microstructure and functions of the skin. (I. Capusan - Cluj

Cloning and complete amino acid sequences of human and murine basement membrane protein BM-40 (SPARC, osteonectin)

AbstractAmino acid sequences of 285 and 286 residues, respectively, were deduced for mouse and human BM-40 from cDNA clones isolated from expression libraries. The sequences showed 92% identity and were also essentially identical to those of bone osteonectin and of the parietal endoderm protein SPARC. About 60% of the mouse BM-40 sequence was confirmed by Edman degradation. Two of the seven disulfide bonds were localized which apparently separate two distinct domains of mouse BM-40

Immunochemistry of Elastotic Material in Sun-Damaged Skin

The nature of elastotic material in sun-damaged human skin was investigated by indirect immunofluorescence. Antibodies were used against the following components of the dermis: type I and type VI collagens, aminopropeptide of type I and type III procollagens, fibronectin, elastin, microfibrillar proteins, and basement membrane represented by the 7S domain of type IV collagen, laminin, and nidogen. The elastotic material exhibited marked fluorescence for elastin and microfibrillar proteins which codistributed with fibronectin. The presence of type I and VI collagens and procollagen type III were demonstrated to a lesser extent within the elastotic material. These results suggest that solar elastosis is primarily derived from elastic fibers and not from preexisting or newly synthesized collagens

Immunohistochemical localization of collagen VI in diabetic glomeruli

Immunohistochemical localization of collagen VI in diabetic glomeruli. Late stage diabetic nephropathy is histologically characterized by either diffuse or nodular expansion of the glomerular matrix. This is presumed to represent the morphological correlate for the functional impairment of the kidney. The exact matrix composition of the nodular glomerulosclerosis lesion of end-stage diabetic nephropathy is not known. Biochemical studies have provided evidence that the microfibrillar collagen type VI is increased in diabetic nephropathy. Consequently, this immunohistochemical study was designed to evaluate the extent and exact morphologic location of increased collagen VI deposition at various stages of diabetic glomerulosclerosis (GS). An irregular, sometimes spot-like staining of collagen VI was observed in diffuse GS in the mesangial portion. The uninterrupted staining which was evident along the glomerular basement membrane in normal glomeruli was discontinuous in diffusely sclerotic glomeruli. In nodular GS, the markedly increased deposition of collagen VI appeared to be evenly distributed throughout the entire nodular lesion. At the same time, mesangial staining for collagen IV was reduced in nodular GS, suggesting that in the expanded mesangial matrix collagen IV is progressively substituted by collagen VI during the transition from diffuse to nodular GS. The colocalization of PAS staining with collagen VI deposition in nodular GS suggests that the typical Kimmelstiel-Wilson lesions at least in part consist of collagen VI. Biochemical analysis confirmed the increased collagen VI deposition in glomeruli extracted from diabetic patients with nodular GS. Application of two antisera, recognizing primarily the α1(VI)- and α2(VI)-chains and the N-terminal part of α3(VI)-chain, respectively, revealed no difference in staining pattern. Comparison of the immunohistochemical results with clinical parameters of diabetic nephropathy suggested that increasing collagen VI deposition may be an indicator of the irreversible remodeling of the glomerular matrix to nodular GS which is associated with functional insufficiency. Our findings indicate striking differences of the mesangial matrix composition in diffuse and nodular GS. These observations together with earlier results provide evidence for a “switch” in the matrix protein production in association with the development of nodular GS in diabetic nephropathy