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R Probstmeier

Publications and source records attributed to R Probstmeier.

34 records · Page 2Linked to original sources
PubMed1996

Structural analysis of the murine cell adhesion molecule L1 by electron microscopy and computer-assisted modelling.

In the present study we have analysed the morphology of two fragments with apparent molecular weights of 180 and 140 kDA (L1-180 and L1-140) derived from the extracellular region of the murine neural cell adhesion molecule L1. The fragment L1-180 consists of almost the entire extracellular part of the molecule, and is built up of six immunoglobulin-like and five fibronectin type III-like domains. Fragment L1-140 lacks one-half of the third, the fourth and the fifth fibronectin type III-like domains. By electron microscopic analysis of rotary-shadowed molecules, L1-140 and L1-180 revealed fibrillar structures 31-43 nm long and 7-12 nm wide with one pronounced globular terminal domain. As determined by complex formation with an L1 antibody, this terminal part of the molecule is formed by the fibronectin type III-like domains. The individual structures showed variation and complexity, and four distinct aspects were identified. These different forms probably represent two-dimensional projections of the same three-dimensional helical structure. Computer-assisted modelling of the L1 molecule, i.e. the protein backbone, showed no strong intramolecular interaction between the different fibronectin type III- or Ig-like domains, suggesting that the formation of the globular part of the molecule is probably achieved by protein-carbohydrate and/or carbohydrate-carbohydrates rather than protein-protein interactions. In addition, our model proposes that interactions occur within the interfaces between the different domains. The highly conserved amino acid residues in these regions point to the necessity of maintaining the orientation between the different domains.

Animals↗
PubMed1995

Functional topography of myelin-associated glycoprotein. II. Mapping of domains on molecular fragments.

The myelin-associated glycoprotein (MAG), an adhesion molecule of the immunoglobulin (Ig) superfamily with five Ig-like domains, was investigated with regard to its binding site(s) for the neuronal cell surface, collagen I, and heparin, using a panel of new monoclonal antibodies and cyanogen bromide cleavage fragments of MAG. All antibodies generated competed with each other for binding to MAG, indicating that they reacted with identical or closely related epitopes. Mapping of the reactive epitopes on recombinant deletion fragments of MAG expressed by Chinese hamster ovary (CHO) fibroblasts showed reactivity of monoclonal antibody 513 with domains I, II, and III, comprising the amino-terminal end of the extracellular domain. Monoclonal antibody 15 recognized domain III only. Binding of MAG-containing liposomes to neurons was blocked by antibodies 15 and 513. Cyanogen bromide (CNBr) fragments of domains I, II, and III bound to collagen type I under isotonic buffer conditions. CNBr fragments containing domains I and II were involved in binding to heparin. These observations suggest that domain III may be important for binding to the neuronal cell surface receptor for MAG, while domains I, II, and III interact with collagen type I and domains II and III with heparin.

3T3 Cells↗
PubMed1995

Recognition molecules myelin-associated glycoprotein and tenascin-C inhibit integrin-mediated adhesion of neural cells to collagen.

Because of the importance of collagens in mediating cell-substrate interactions and the association of collagens with neural recognition molecules in the peripheral nervous system, the ability of neural recognition molecules to modify the substrate properties of collagens, in particular collagen type I, for cell adhesion was determined. Two cell lines, the N2A neuroblastoma and PC12 pheochromocytoma, were investigated for their capacity to adhere to different collagen types in the absence or presence of several neural recognition molecules. Adhesion of N2A or PC12 cells and membrane vesicles from PC12 cells to collagen type I was reduced when the collagen had been preincubated prior to its application as substrate with the extracellular domain of myelin-associated glycoprotein (s-MAG) or, as control, fibroblast tenascin-C (F-tenascin). In mixture with other collagen types, s-MAG was only able to reduce the adhesiveness of collagen types III and V, but not of collagen types II and IV. F-tenascin reduced the adhesiveness of all collagen types tested. In contrast to F-tenascin, s-MAG had to be present during fibrillogenesis to exert its effect, indicating that it must be coassembled into the collagen fibril to block the binding site. Cell adhesion to collagen type I was dependent on Mg2+ or Mn2+ and inhibited by a monoclonal antibody to the alpha 1 integrin subunit. The combined observations indicate that s-MAG and F-tenascin interfere with cell binding, most probably by modifying the integrin binding site, and that the two molecules act by different mechanisms, both leading to reduction of adhesion.

Animals↗
PubMed1995

Galectin-3, a beta-galactoside-binding animal lectin, binds to neural recognition molecules.

In this study, we have investigated the ability of galectin-3, a beta-galactoside-binding animal lectin, to interact in vitro with different neural tissue-derived glycoproteins involved in cell-cell and cell-substrate adhesion. Galectin-3 interacted to varying degrees with the cell recognition molecules L1, the myelin-associated glycoprotein, and the neural cell adhesion molecule and the extracellular matrix molecules tenascin-C and tenascin-R but not with collagen type I. Binding of galectin-3 to the different glycoproteins tested was carbohydrate dependent and could be specifically inhibited by the addition of lactose and, to a lesser extent, galactose.

Animals↗
PubMed1994

Expression of the neural cell adhesion molecule and polysialic acid during early mouse embryogenesis.

The expression of the neural cell adhesion molecule (N-CAM) and alpha 2-8 linked polysialic acid (PSA), which is believed to be predominantly expressed on N-CAM, was investigated during early embryonic development of the mouse (embryonic days 7.5 to 10.0). By immunocytochemistry, in tissue sections, N-CAM and PSA were not detectable at embryonic day 7.5 but were expressed in the prominent body regions such as somites, unsegmented mesoderm, developing heart, and neuroectoderm at embryonic day 8.0 N-CAM and PSA immunoreactivities were always predominantly associated with the plasma membrane. No tissue could be detected which was positive for PSA but negative for N-CAM. In Western blot analysis of whole embryos, by contrast, only the lightly sialylated and PSA-negative 180 and 140 kD isoforms of N-CAM were present at embryonic day 8.0 and strong expression of PSA-bearing, heavily sialylated N-CAM was not detectable before embryonic day 10.0. In Western blot analysis of N-CAM immunoaffinity purified from whole embryos and digested with neuraminidase as well as in Northern blot analysis, the 120 kD isoform of N-CAM or its corresponding mRNA were not expressed in detectable amounts during the time period investigated.

Animals↗
PubMed1994

Tenascin-R (J1 160/180 inhibits fibronectin-mediated cell adhesion--functional relatedness to tenascin-C.

Cell adhesion and neurite outgrowth on fibronectin is a multistep process modulated by different extra- and intracellular signals. Fibronectin-mediated cell attachment and spreading can be affected in a negative way by tenascin-C, an extracellular matrix glycoprotein expressed in a temporally and spacially restricted manner during early morphogenesis. Tenascin-R (J1-160/180), consisting of two major isoforms of 160 kDa (tenascin-R 160) and 180 kDa (tenascin-R 180) in mammals, is an extracellular matrix glycoprotein of the central nervous system that shares high structural homologies with tenascin-C. Here we show that in relation to fibronectin-mediated adhesion, the two extracellular matrix molecules are also functionally closely related. When offered as mixed substrata with other extracellular matrix molecules, the two tenascin-R isoforms and tenascin-C derived from mouse brain selectively inhibit fibronectin-dependent cell adhesion and neurite outgrowth, and affect cell morphology of different mesenchymal and neural cells. This effect is partially due to interactions at the substrate level that result in a steric hindrance and/or conformational change of the cell binding sites of the fibronectin molecule. In addition, tenascin-R 180 and tenascin-C interact with cells by an RGD- and beta 1 integrin-independent mechanism, leading to cell rounding and detachment from such substrata. The expression of tenascin-R and tenascin-C in the nervous system at times and locations where fibronectin-mediated cellular processes take place may be related to the role of inhibitory signals in the extracellular matrix in the regulation of cell migration and differentiation in general.

Animals↗
PubMed1993

Functional topography of the myelin-associated glycoprotein. I. Mapping of domains by electron microscopy.

The functional topography of the myelin-associated glycoprotein (MAG) was investigated by electron microscopic analysis of rotary-shadowed molecules of a MAG fragment (MAG 90) comprising the five immunoglobulin-like domains of the extracellular part of the molecule. MAG 90 molecules appeared as rod-like structures (18.5 +/- 1.2 nm long and 4.0 +/- 0.8 nm wide) with a globular domain at one end. Antibodies directed against the amino- and carboxy-terminus of MAG 90 interacted with the non-globular terminal region, indicating that the molecule is bent in the globular region with the amino- and carboxy-terminal arms in close apposition to each other. An antibody which interferes with the binding of MAG to neurons interacted predominantly with the globular domain of MAG 90. The fibril-forming collagen types I, III and V bound mainly to the non-globular terminal region of MAG 90, whereas the majority of heparin molecules interacted with the globular region of the molecule. The L2/HNK-1 carbohydrate structure was localized at the non-globular region in the protein fragment comprising the fourth and fifth immunoglobulin-like domains.

Animals↗
PubMed1992

Interactions of the neural cell adhesion molecule and the myelin-associated glycoprotein with collagen type I: involvement in fibrillogenesis.

To gain insights into the functional role of the molecular association between neural adhesion molecules and extracellular matrix constituents, soluble forms of the myelin-associated glycoprotein (MAG) and the neural cell adhesion molecule (N-CAM), representing most of the extracellular domains of the molecules, were investigated in their ability to modify fibrillogenesis of collagen type I. MAG and N-CAM retarded the rate of fibril formation, as measured by changes in turbidity, and increased the diameter of the fibrils formed, but did not change the banding pattern when compared to collagen type I in the absence of adhesion molecules. Scatchard plot analysis of the binding of MAG and N-CAM to the fibril-forming collagen types I, II, III, and V suggest one binding site for N-CAM and two binding sites for MAG. Binding of MAG, but not of N-CAM, to collagen type I was decreased during fibril formation, probably due to a reduced accessibility of one binding site for MAG during fibrillogenesis. These results indicate that the neural adhesion molecules can influence the configuration of extracellular matrix constituents, thus, implicating them in the modulation of cell-substrate interactions.

Animals↗
PubMed1990

Expression of the adhesion molecules L1, N-CAM and J1/tenascin during development of the murine small intestine.

We have previously studied the immunohistological localization of the three adhesion molecules L1, N-CAM and J1/tenascin in adult mouse small intestine and shown that L1 expression in epithelial crypt cells underlies the adhesion of these cells to one another [63]. To obtain further insight into the functional roles of L1, N-CAM and J1/tenascin in this organ we studied their expression starting at embryonic day 14 during embryonic and early postnatal morphogenesis and during epithelial cell migration in the adult. Expression of L1 was restricted to neural cells until approximately postnatal day 5, when L1 started to be detectable on crypt but not on villus cells, predominantly on the basolateral membrane infoldings. As in brain, L1-specific mRNA was approximately 6 kb in size. L1 from intestine appears to differ from the brain-derived equivalent in possessing a higher level of glycosylation. N-CAM was detectable from embryonic day 14 onward in neural and also in mesenchymal cells. Expression by smooth muscle cells decreased during development. In the villus core, N-CAM was strongly detectable at contact sites between smooth muscle cells forming the cellular scaffold of the villus. From embryonic day 14 onward, N-CAM appeared in both 180- and 140-kDa forms. J1/tenascin was present in both neural and mesenchymal cells from embryonic day 14 onward. Starting at embryonic day 17, J1/tenascin appeared concentrated at the boundary between mesenchyme and epithelium in an increasing gradient from the crypt base to the villus top. From embryonic day 14 onward J1/tenascin consisted of the 190- and 220-kDa components. J1/tenascin from intestine differed from brain-derived J1 in its carbohydrate composition. These observations show that the three adhesion molecules are expressed by distinct cell populations and may serve as cell-type-specific markers in pathologically altered intestinal tissue.

Animals↗
PubMed1990

Fibroblast-derived J1 adhesion glycoproteins show binding properties to extracellular matrix constituents different from those of central nervous system origin.

The J1 extracellular adhesion molecule from mouse brain consists of several immunochemically related glycoproteins of different molecular weights and distinct functional properties. Like the brain J1 glycoproteins, the fibroblast-derived J1 glycoproteins interact with all collagen types tested (collagen G and types I-IV and IX), as measured by binding of 125I-labeled J1 glycoproteins to immobilized collagens. As tested for collagen type I, this binding can be inhibited more effectively by chondroitin sulfate than by heparin. After electrophoretic separation and transfer to nitrocellulose, fibroblast-derived J1 only binds to a limited number of collagen types (collagen types I, VI, and IX and G), whereas brain-derived J1 glycoproteins bind to all collagen types tested (collagen types I-VI and IX and G). These results show that fibroblast-derived J1 glycoproteins, although immunochemically related to J1 glycoproteins from brain, differ from these in their binding specificities to extracellular matrix constituents.

Animals↗
PubMed1990

Expression of J1/tenascin in the crypt-villus unit of adult mouse small intestine: implications for its role in epithelial cell shedding.

The localization of the extracellular matrix recognition molecule J1/tenascin was investigated in the crypt-villus unit of the adult mouse ileum by immunoelectron microscopic techniques. In the villus region, J1/tenascin was detected strongly in the extracellular matrix (ECM) between fibroblasts of the lamina propria. It was generally absent in the ECM at the interface between subepithelial fibroblasts and intestinal epithelium, except for some restricted areas along the epithelial basal lamina of villi, but not of crypts. These restricted areas corresponded approximately to the basal part of one epithelial cell. In J1/tenascin-positive areas, epithelial cells contacted the basal lamina with numerous microvillus-like processes, whereas in J1/tenascin-negative areas the basal surface membranes of epithelial cells contacted their basal lamina in a smooth and continuous apposition. In order to characterize the functional role of J1/tenascin in the interaction between epithelial cells and ECM, the intestinal epithelial cell line HT-29 was tested for its ability to adhere to different ECM components. Cells adhered to substratum-immobilized fibronectin, laminin and collagen types I to IV, but not to J1/tenascin. When laminin or collagen types I to IV were mixed with J1/tenascin, cell adhesion was as effective as without J1/tenascin. However, adhesion was completely abolished when cells were offered a mixture of fibronectin and J1/tenascin as substratum. The ability of J1/tenascin to reduce the adhesion of intestinal epithelial cells to their fibronectin-containing basal lamina suggests that J1/tenascin may be involved in the process of physiological cell shedding from the villus.

Animals↗
PubMed1989

Binding properties of the neural cell adhesion molecule to different components of the extracellular matrix.

A soluble form of the neural cell adhesion molecule (N-CAM) was obtained from 100,000-g supernatants of crude brain membrane fractions by incubation for 2 h at 37 degrees C. The isolated N-CAM, consisting of one polypeptide chain with a molecular mass of 110 kilodaltons (N-CAM 110), was studied for its binding specificity to different components of the extracellular matrix (ECM). N-CAM 110 bound to different types of collagen (collagen types I-VI and IX). The binding efficiency was dependent on salt concentration and could be called specific according to the following criteria: (a) Binding showed substrate specificity (binding to collagens, but not to other ECM components, such as laminin or fibronectin). (b) Binding of N-CAM 110 to heat-denatured collagens was absent or substantially reduced. (c) Binding was saturable (Scatchard plot analyses were linear with KD values in the range of 9.3-2.0 X 10(-9) M, depending on the collagen type and buffer conditions). Binding of N-CAM 110 to collagens could be prevented in a concentration-dependent manner by the glycosaminoglycans heparin and chondroitin sulfate. N-CAM 110 also interacted with immobilized heparin, and this interaction could be prevented by heparin and chondroitin sulfate. Thus, in addition to its role in cell-cell adhesion, N-CAM is a binding partner for different ECM components, an observation suggesting that it also serves as a substrate adhesion molecule in vivo.

Animals↗
PubMed1987

Characterization of the cell adhesion molecules L1, N-CAM and J1 in the mouse intestine.

To gain insight into the cellular and molecular mechanisms underlying epithelial cell surface interactions in the adult mouse intestine, we have characterized the cell adhesion molecules L1, N-CAM and J1 by immunocytological, biochemical and cell biological methods. Whereas N-CAM and J1 expression was found to be confined to the mesenchymal and neuroectodermally-derived parts of the intestine, L1 was localized in the proliferating epithelial progenitor cells of crypts, but not in the more differentiated epithelial cells of villi. L1 was detected in crypt cells by Western blot analysis in the molecular forms characteristic of peripheral neural cells, with apparent mol. wts of 230, 180 and 150 kd. Aggregation of single, enriched crypt, but not villus cells, was strongly inhibited in the presence of Fab fragments of polyclonal L1 antibodies. These observations show that L1 is not confined to the nervous system and that it may play a functional role in the histogenesis of the intestine in the adult animal.

Animals↗
PubMed1986

Epidermal growth factor is not detectable in developing and adult rodent brain by a sensitive double-site enzyme immunoassay.

A highly sensitive double-site enzyme immunoassay for epidermal growth factor (EGF) was used to quantify EGF concentrations in brain and cerebrospinal fluid of early postnatal and adult mice and rats. EGF was not detectable under any condition at sensitivity levels of 0.06 ng/g wet wt. These observations support the notion that EGF receptors on astrocytes are triggered by other growth factors than EGF.

Animals↗
PubMed1985

Epidermal growth factor does not cross the blood-brain barrier.

To measure the passage of epidermal growth factor (EGF) through the blood-brain barrier (BBB) 125I-labeled EGF was injected intravenously into adult rats. The distribution of 125I-EGF in the blood and cerebrospinal fluid (CSF) was determined over a time period of several hours. Between 2 to 6 h a stable distribution of intact 125I-EGF in CSF was measured to be approximately 1/500 of the blood-borne EGF, an equilibrium value below those obtained by other investigators for BBB-impermeable compounds, such as inulin and bovine serum albumin. Our data indicate that 125I-EGF, although clearly detectable in the CSF, does not cross the BBB at a higher rate or in higher quantities than would be expected from its molecular size.

Animals↗