http://www.jbc.org/content/264/27/16174.full.pdf

A synthetic peptide containing the IKVAV sequence from the A chain of laminin mediates cell attachment, migration, and neurite outgrowth.

Haematopoietic progenitor cells proliferate and mature in semi-solid media when stimulated by exogenous haematopoietic cell growth factors (HCGFs) such as granulocyte-macrophage colony-stimulating factor (GM-CSF)1,2. They also proliferate in association with marrow-derived stromal cells3,4 although biologically active amounts of HCGFs cannot be detected in stromal culture supernatants5. It is possible that HCGFs are synthesized in small amounts by stromal cells but remain bound to the stromal cells and/or their extracellular matrix (ECM). This interpretation accords with haematopoietic progenitor cell proliferation in close association with stromal layers in long-term cultures6. Glycosaminoglycans (GAGs) are found in the ECM produced by stromal cells27,8. They are prime candidates for selectively retaining HCGFs in the stromal layer9; they influence embryonic morphogenesis and cyto-differentiation10 and they may regulate haematopoiesis11–13. We now report that granulocyte-macrophage colony-stimulating activity can be eluted from cultured stromal layers and that exogenous GM-CSF binds to GAGs from bone marrow stromal ECM. Selective Compartmentalization of HCGFs in this manner may be an important function of the marrow microenvironment and may be involved in haematopoietic cell regulation.

Compartmentalization of a haematopoietic growth factor (GM-CSF) by glycosaminoglycans in the bone marrow microenvironment

Structure and function of heparan sulphate proteoglycans

Laminin is a multifunctional protein with diverse biological activities. Like fibronectin, it can influence cell adhesion, growth, morphology, differentiation, migration, and agglutination as well as the assembly of the extracellular matrix. Laminin primarily affects cells of epithelial origin, and the response varies depending on the cell. Because most differentiated cells are difficult to maintain in culture, laminin may be an important supplement in studies on cell differentiation in vitro.

Biological activities of laminin

Publisher Summary This chapter describes the structure and biological activity of heparin. Being an alternating copolymer of a hexosamine and an alduronic acid, heparin belongs to the class of glycosaminoglycans (GAG). The repeating disaccharide units constituting the building-blocks that account for the largest fraction of the most common GAG are elaborated in the chapter. Heparin is most commonly prepared as the sodium salt that can be converted into other salts by cation exchange. The final yield and purity of a heparin preparation depend largely on the use of appropriate, analytical methods at different stages of extraction and purification. Heparin in the mixtures of glycosaminoglycans can also be titrated to a turbidimetric end-point with quaternary ammonium salts, such as cetyltrimethylpyridinium chloride (CPC), at a sufficiently high concentration of salt to prevent complex-formation by less-charged polyanions. The structure of the main type of hexosamine residue in heparin—that is, 2-deoxy-2-sulfoamino- d -glucose 6-sulfate, was established by early studies. Crystalline 2-amino-2-deoxy- d -glucose hydrochloride was isolated in high yield from acid hydrolyzates of the polysaccharide and its structure was confirmed by oxidation to 2-amino-2-deoxy- d -gluconic acid, Q2 and by degradation with ninhydrin. The methods for controlled depolymerization of heparin are also presented in the chapter.

Structure and biological activity of heparin

The binding of the basement-membrane glycoprotein laminin to glycosaminoglycans (aggregating and non-aggregating subsets of heparan sulphates and dermatan sulphates, as well as heparin, chondroitin sulphates and hyaluronic acid) was studied by affinity chromatography. Partially periodate-oxidized chains of glycosaminoglycans were coupled to adipic acid dihydrazide-substituted agarose. Co-polymeric glycosaminoglycans reveal high affinity for laminin, whereas hyaluronic acid does not. Competitive-release experiments indicate that glycosaminoglycans share a common binding site on the laminin molecule.

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Binding of the basement-membrane glycoprotein laminin to glycosaminoglycans. An affinity-chromatography study.

Involvement of glycosaminoglycans in detachment of early myeloid precusors from bone-marrow stromal cells

Point-mutated p21ras couples a muscarinic receptor to calcium channels and polyphosphoinositide hydrolysis.

In a series of attempts to reveal plasma heparin, we found that high ionic strength and modification of protein amino groups were not effective in extracting endogenous heparin (or, indeed, a large percentage of exogenous labelled heparin), whereas delipidation in the presence of 4M-guanidinium chloride gave high yields, indicating that plasma heparin may be assembled with compounds other than proteins, in a form making it inaccessible to water and ions. During the extraction of lipids, a paradoxical entry of heparin into the organic phase was observed. Detergents, including sodium dodecyl sulphate, did not shift heparin into the aqueous phase, whereas repeated chloroform/methanol extraction did so. Using purified compounds we were able to reproduce in vitro both the scavenging of heparin from water as well as the formation of heparin-phosphatidylcholine complexes soluble in organic solvents. Evidence for complexing of heparin with phosphatidylcholine was also obtained by electrophoretic and ultracentrifugation assays. The quaternary-ammonium-containing phosphatidylcholine was the more effective phospholipid in binding heparin; anionic phospholipids did not bind. Only heparin-like glycosaminoglycans bound phosphatidylcholine, but less-sulphated compounds (heparan sulphate and dermatan sulphate) were weaker ligands. Gel-filtration experiments showed that heparin was not bound to liposome vesicles, but that a measurable percentage of the phospholipids was stripped off from vesicles and was found in the form of a complex separable from liposomes by gel filtration. The molecular basis as well as the biological role of the interaction of heparin with major membrane phospholipids are discussed.

Complexing of heparin with phosphatidylcholine. A possible supramolecular assembly of plasma heparin.

Resting 3T3 cells have relatively more sulphated glycosaminoglycans and Ca2+ in their cell coat than do growing or SV40-virus-transformed cells. It is suggested that the different Ca2+-binding capacity controls cellular activities by affecting the distribution of Ca2+ between the intra- and ecto-cellular compartments.

/pdf/surface-glycosaminoglycans-and-calcium-distribution-in-3t3-h1ghhzlsb7.pdf

S Vannucchi

Papers

Binding of the basement-membrane glycoprotein laminin to glycosaminoglycans. An affinity-chromatography study.

Involvement of glycosaminoglycans in detachment of early myeloid precusors from bone-marrow stromal cells

Point-mutated p21ras couples a muscarinic receptor to calcium channels and polyphosphoinositide hydrolysis.

Complexing of heparin with phosphatidylcholine. A possible supramolecular assembly of plasma heparin.

Surface glycosaminoglycans and calcium distribution in 3T3 cells