Heparan sulfate and control of cell division: adhesion and proliferation of mutant CHO-745 cells lacking xylosyl transferase

We have examined the role of cell surface glycosaminoglycans in cell division: adhesion and proliferation of Chinese hamster ovary (CHO) cells. We used both wild-type (CHO-K1) cells and a mutant (CHO-745) which is deficient in the synthesis of proteoglycans due to lack of activity of xylosyl transferase. Using different amounts of wild-type and mutant cells, little adhesion was observed in the presence of laminin and type I collagen. However, when fibronectin or vitronectin was used as substrate, there was an enhancement in the adhesion of wild-type and mutant cells. Only CHO-K1 cells showed a time-dependent adhesion on type IV collagen. These results suggest that the two cell lines present different adhesive profiles. Several lines of experimental evidence suggest that heparan sulfate proteoglycans play a role in cell adhesion as positive modulators of cell proliferation and as key participants in the process of cell division. Proliferation and cell cycle assays clearly demonstrate that a decrease in the amount of glycosaminoglycans does not inhibit the proliferation of mutant CHO-745 cells when compared to the wild type CHO-K1, in agreement with the findings that both CHO-K1 and CHO-745 cells take 8 h to enter the S phase

Extracellular matrix molecules as targets for brown spider venom toxins

Loxoscelism, the term used to describe lesions and clinical manifestations induced by brown spider's venom (Loxosceles genus), has attracted much attention over the last years. Brown spider bites have been reported to cause a local and acute inflammatory reaction that may evolve to dermonecrosis (a hallmark of envenomation) and hemorrhage at the bite site, besides systemic manifestations such as thrombocytopenia, disseminated intravascular coagulation, hemolysis, and renal failure. The molecular mechanisms by which Loxosceles venoms induce injury are currently under investigation. In this review, we focused on the latest reports describing the biological and physiopathological aspects of loxoscelism, with reference mainly to the proteases recently described as metalloproteases and serine proteases, as well as on the proteolytic effects triggered by L. intermedia venom upon extracellular matrix constituents such as fibronectin, fibrinogen, entactin and heparan sulfate proteoglycan, besides the disruptive activity of the venom on Engelbreth-Holm-Swarm basement membranes. Degradation of these extracellular matrix molecules and the observed disruption of basement membranes could be related to deleterious activities of the venom such as loss of vessel and glomerular integrity and spreading of the venom toxins to underlying tissues

A fucan from the brown seaweed Spatoglossum schröederi inhibits Chinese hamster ovary cell adhesion to several extracellular matrix proteins

Fucans, a family of sulfated polysaccharides present in brown seaweed, have several biological activities. Their use as drugs would offer the advantage of no potential risk of contamination with viruses or particles such as prions. A fucan prepared from Spatoglossum schröederi was tested as a possible inhibitor of cell-matrix interactions using wild-type Chinese hamster ovary cells (CHO-K1) and the mutant type deficient in xylosyltransferase (CHO-745). The effect of this polymer on adhesion properties with specific extracellular matrix components was studied using several matrix proteins as substrates for cell attachment. Treatment with the polymer inhibited the adhesion of fibronectin to both CHO-K1 (2 x 10(5))()and CHO-745 (2 x 10(5) and 5 x 10(5)) cells. No effect was detected with laminin, using the two cell types. On the other hand, adhesion to vitronectin was inhibited in CHO-K1 cells and adhesion to type I collagen was inhibited in CHO-745 cells. In spite of this inhibition, the fucan did not affect either cell proliferation or cell cycle. These results demonstrate that this polymer is a new anti-adhesive compound with potential pharmacological applications

Detection of post-translational sulfation of alpha-5 beta-1 integrin and its role in integrin-fibronectin binding

Fibronectins are glycoproteins of the extracellular matrix composed of two 220-kDa polypeptide chains named A and B bound by two disulfide bridges. Both chains when digested with proteolytic enzymes give rise to six different domains named I to VI that are involved in the ligand properties of this molecule. Fibronectins bind fibrin, collagen, glycosaminoglycan residues and several integrins. In this study, using metabolic radiolabeling of alpha(5)beta(1) integrin with sodium sulfate, an immunoprecipitation reaction, inhibition of sulfate incorporation an a fibronectin-binding assay, we were able to detect this integrin as a sulfated molecule and this sulfation appears to regulate the integrin-fibronectin binding.

Cellular adhesion to laminin involves a chondroitin sulfate proteoglycan

Cell-extracellular matrix interactions are intimately involved in the regulation of many cellular processes such as embryonic development or tumor cell growth and metastasis. In our previous work we were able to detect a 90/100-kDa laminin binding chondroitin sulfate proteoglycan. A search for this molecule in different cell lines showed that it is only found in cells that adhere to laminin.

Trypanosoma cruzi binds to laminin in a carbohydrate-independent way

The bindings of 125I-laminin to trypomastigotes is specific and 2-5 x 10**3 laminin-binding sites were calculated to be presented on the surface of a live trypomastigote. Anti-laminin antibodies were able to inhibit the invasion of cultured cells by trypomastigotes (62-75 per cent), suggesting that laminin may be involved in the adhesion of the parasite to host cells. By affinity chromatography, an 85-KDa glycoprotein was isolated (laminin-bindign glycoprotein, LBG) from trypomastigote lysates, but not from epimastigote lysates. It is suggested that at least fragment E8 (but not E1) from laminin could be involbed in the reaction which is independent of the carbohydrate moieties from both ligand and recepto. It is also shown that LBG is member of the Tc-85 family, previously shown to be related to the invasion process of the parasite

Identification and characterization of highly conserved antigenic determinants in the laminin molecule

1. Fragments P1 and E8, the results of two different enzymatic digestions of the laminin molecule, represent interaction sites of laminin with specific. By using negative and positive affinity purification of a rabbit antiserum against mouse laminin we have generated antibodies to these two fragments. 2. Antibodies against P1 were able to immunoprecipitate fragment E8 from elastase-digested laminin. By liquid phase competition experiments we demonstrated that the epitopes shared by P1 and E8 are a minor portion of the antigenic determinants of P1. When we checked for the presence of these shared epitopes in the human laminin molecule, they were the major fraction of the interspecies antigenic conservation. 3. A similar approach usisng polyclonal antibodies against human laminin has confirmed these reults. 4. The shared epitopes present in both mouse and human laminin molecules seem to be spatially determined, because antibodies against these sites did not bind to fully denatured laminin. 5. Since human and mouse laminin bind to cell receptors and to other extracellular matrix proteins from both species, we conclude that these antigenic determinants may represent the actual sites for at least some of these interactions