Collagen V is a minor component of the heterotypic I/III/V collagen fibrils and the defective product in most cases of classical Ehlers Danlos syndrome (EDS). The present study was undertaken to elucidate the impact of collagen V mutations on skin development, the most severely affected EDS tissues, using mice harboring a targeted deletion of the ␣2(V) collagen gene (Col5a2). Contrary to the original report, our studies indicate that the Col5a2 deletion (a.k.a. the pN allele) represents a functionally null mutation that affects matrix assembly through a complex sequence of events. First the mutation impairs assembly and/or secretion of the ␣1(V) 2 ␣2(V) heterotrimer with the result that the ␣1(V) homotrimer is the predominant species deposited into the matrix. Second, the ␣1(V) homotrimer is excluded from incorporation into the heterotypic collagen fibrils and this in turn severely impairs matrix organization. Third, the mutant matrix stimulates a compensatory loop by the ␣1(V) collagen gene that leads to additional deposition of ␣1(V) homotrimers. These data therefore underscore the importance of the collagen V heterotrimer in dermal fibrillogenesis. Furthermore, reduced thickness of the basement membranes underlying the epidermis and increased apoptosis of the stromal fibroblasts in pN/pN skin strongly indicate additional roles of collagen V in the development of a functional skin matrix.The physiological and biomechanical properties of different extracellular matrices depend in large part on the timely and tissue-specific deposition of collagen trimers (or types) that assemble into unique macromolecular aggregates. Among them, the banded fibrils represent the most abundant and widely distributed class of collagen assemblies. Fibril-forming collagens include five distinct types (I to III, V, and XI), which are found in most connective tissues (I, III, and V) or only in cartilage and vitreous (II and XI). Genetic studies have underscored the critical contribution of fibrillar collagens to organismal function by correlating mutations in the ␣ chain subunits with the genesis of several connective tissue disorders (17). Relevant to the present study, they have established the role of collagen V in regulating collagen I fibrillogenesis and in maintaining tissue integrity.Collagen V is a quantitatively minor component of tissues rich in collagen I, such as dermis, tendons/ligaments, bones, blood vessels, and cornea. Unlike other tissues, where collagen V represents only 1 to 3% of the total collagen fiber content, the relative concentration of this collagen type in cornea is significantly higher, 20 to 25% (3). Collagen V copolymerizes with collagens I and III to form heterotypic I/III/V fibrils in which the triple helical portion of the molecule is embedded and the amino-terminal globular domain projects onto the surface (4, 19). Very thin (5 to 10 nm in diameter) fibrils of collagen V have also been reported immediately near basement membranes and extending into the adjacent interstitial matrix (5,12,14,24). There...

P-glycoprotein (P-gp) is the most well-known ATP-binding cassette (ABC) transporter involved in unidirectional substrate translocation across the membrane lipid bilayer, thereby causing the typical multidrug resistance (MDR) phenotype expressed in many cancers. We observed that in human CEM acute lymphoblastic leukemia cells expressing various degrees of chemoresistance and where P-gp was the sole MDR-related ABC transporter detected, the amount of esterified cholesterol increased linearly with the level of resistance to vinblastine while the amounts of total and free cholesterol increased in a nonlinear way. Membrane cholesterol controlled the ATPase activity of P-gp in a linear manner, whereas the P-gp-induced daunomycin efflux decreased nonlinearly with the depletion of membrane cholesterol. All these elements suggest that cholesterol controls both the ATPase and the drug efflux activities of P-gp. In addition, in CEM cell lines that expressed increasing levels of elevated chemoresistance, the amount of P-gp increases to a plateau value of 40% of the total membrane proteins and remained unvaried while the amount of membrane cholesterol increased with the elevation of the MDR level, strongly suggesting that cholesterol may be directly involved in the typical MDR phenotype. Finally, we showed that the decreased daunomycin efflux by P-gp due to the partial depletion of membrane cholesterol was responsible for the efficient chemosensitization of resistant CEM cells, which could be totally reversed after cholesterol repletion.

Collagen V is a minor component of connective tissues that plays a fundamental role in matrix organization. Indeed, direct evidence was obtained from collagen V gene mutations that provoke obvious alteration of fibril aggregates (1-4). Aside from its role in collagen fibril formation, collagen V interacts specifically with a variety of macromolecules in the extracellular matrix (5) and with several cell-surface receptors such as integrins (6, 7), tyrosine kinase receptors (8, 9), and proteoglycans (10, 11). Such interactions are important in regulating cell behavior and fibril formation during development and physiological events. Although collagen V is involved in a plethora of specific interactions and thus might possess various domainspecific functions, binding sites have not yet been mapped except for the heparin-binding site. Heparin is abundant in animal tissues in the form of heparan sulfate proteoglycans both on the cell surface and in the extracellular matrix. A 30-kDa heparin-binding fragment of the ␣1(V) chain has been isolated and was shown to bind heparin with the same affinity as the complete parental chain (12). The binding site is exclusive to this chain since the two other chains, viz. ␣2(V) and ␣3(V), which can also be part of collagen V molecules, have no affinity for heparin (11-13). Using a recombinant approach, we have narrowed this region down to a 12-kDa fragment referred to as HepV. Interestingly, the recombinant fragment HepV expressed in Escherichia coli was shown to support heparindependent cell adhesion (11).Heparin-binding sites are found in a wide range of proteins including collagens and in a broad repertoire of extracellular matrix proteins, viz. fibronectin, tenascin, and laminin. They are all characterized by an overall positive charge, and common structural motifs have been proposed from the analysis of the different heparin-binding site primary sequences. The consensus sequences BBXB, XBBXBX, and XBBBXXBX (where B designates a basic amino acid and X designates any other residues) have been identified (14). A thorough inspection of the ␣1(V) chain primary sequence indicates that no sequence matches any of the proposed motifs above. HepV does contain a stretch of cationic amino acids (Arg 900 -Arg 924 ), but a synthetic peptide that was designed to encompass this sequence was shown to have negligible affinity for immobilized heparin under physiological conditions. Also, the synthetic peptide failed to compete with HepV or with the complete ␣1(V) chain in a heparin binding assay. This result led to the hypothesis that the peptide conformation or/and the flanking residues might be crucial for efficient binding to heparin (11). The characteristics of the collagen V heparin-binding site might be somehow more subtle than previously thought. Based on the fact that recombinant HepV binds to heparin with the same relative affinity as the parental ␣1(V) chain, all 7 basic residues contained in the region Arg 900 -Arg 924 were individually mutated, and the resulting expression products w...

A minicollagen containing the COL1 and NC1 domains of chicken collagen XII has been produced in insect cells. Significant amounts of trimers contain a triplehelical domain in which the cysteines are not involved in inter-but in intrachain bonds. In reducing conditions, providing that the triple-helix is maintained, disulfide exchange between intra-and interchain bonding is observed, suggesting that the triple-helix forms first and that in favorable redox conditions interchain bonding occurs to stabilize the molecule. This hypothesis is verified by in vitro reassociation studies performed in the presence of reducing agents, demonstrating that the formation of interchain disulfide bonds is not a prerequisite to the trimeric association and triple-helical folding of the collagen XII molecule. Shortening the COL1 domain of minicollagen XII to its five C-terminal GXY triplets results in an absence of trimers. This can be explained by the presence of a collagenous domain that is too short to form a stable triple-helix. In contrast, the presence of five additional C-terminal triplets in COL1 allows the formation of triple-helical disulfide-bonded trimers, suggesting that the presence of a triple-helix is essential for the assembly of collagen XII.Collagens are the most abundant proteins of the extracellular matrix. They consist of collagenous triple-helical domains, flanked by non-collagenous domains, that differ in their size and number from one collagen to another. Collagens can be homotrimers or heterotrimers, and their structure is stabilized by interchain disulfide bonds (1, 2).Collagens IX, XII, XIV, XVI, and XIX constitute the FACITs 1 family that are grouped on the basis of similarities in their short C-terminal domain COL1, which is thought to be important for the association with fibrils (3, 4). The association of collagen type IX, XII, and XIV molecules with quarter-staggered fibrils has been shown by immunofluorescence and electron microscopy (5-9). The biological functions of the FACITsare not yet fully understood. Rapid and reversible production of collagen XII by fibroblasts submitted to mechanical stress has been observed (10, 11). Collagens XII and XIV potentially facilitate the contraction of collagen gels by fibroblasts (12), and they are known to interact with other components of the extracellular matrix (13)(14)(15). This suggests that they contribute to the cohesion and the modulation of the mechanical properties of the extracellular matrix. Collagen XIV is also a potential reservoir for procollagen N-proteinase (16) and promotes the adhesion of different cells (17, 18). The assembly of fibril-forming collagens (collagen types I, II, III, V, and XI) is a complex process in which three chains must fold into a triple-helix. This results from chain association, nucleation, and propagation of the folding occurring from the C to N termini in a zipper-like fashion (19,20). The C-terminal non-triple-helical domains, called the C-propeptides, play an important role in the initial steps of chain associatio...