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

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

Expression of ECM proteins fibulin-1 and -2 in acute and chronic liver disease and in cultured rat liver cells

Fibulin-2 has previously been considered as a marker to distinguish rat liver myofibroblasts from hepatic stellate cells. The function of other fibulins in acute or chronic liver damage has not yet been investigated. The aim of this study has been to evaluate the expression of fibulin-1 and -2 in models of rat liver injury and in human liver cirrhosis. Their cellular sources have also been investigated. In normal rat liver, fibulin-1 and -2 were both mainly present in the portal field. Fibulin-1-coding transcripts were detected in total RNA of normal rat liver, whereas fibulin-2 mRNA was only detected by sensitive, real-time quantitative polymerase chain reaction. In acute liver injury, the expression of fibulin-1 was significantly increased (17.23-fold after 48 h), whereas that of fibulin-2 was not modified. The expression of both fibulin-1 and -2 was increased in experimental rat liver cirrhosis (19.16- and 26.47-fold, respectively). At the cellular level, fibulin-1 was detectable in hepatocytes, “activated” hepatic stellate cells, and liver myofibroblasts (2.71-, 122.65-, and 469.48-fold over the expression in normal rat liver), whereas fibulin-2 was restricted to liver myofibroblasts and was regulated by transforming growth factor beta-1 (TGF-β1) in 2-day-old hepatocyte cultures and in liver myofibroblasts. Thus, fibulin-1 and -2 respond differentially to single and repeated damaging noxae, and their expression is differently present in liver cells. Expression of the fibulin-2 gene is regulated by TGF-β1 in liver myofibroblasts