Olga Bondar

Type IV Collagen of the Glomerular Basement Membrane EVIDENCE THAT THE CHAIN SPECIFICITY OF NETWORK ASSEMBLY IS ENCODED BY THE NONCOLLAGENOUS NC1 DOMAINS

The ultrafiltration function of the glomerular basement membrane (GBM) of the kidney is impaired in genetic and acquired diseases that affect type IV collagen. The GBM is composed of five (α1 to α5) of the six chains of type IV collagen, organized into an α1·α2(IV) and an α3·α4·α5(IV) network. In Alport syndrome, mutations in any of the genes encoding the α3(IV), α4(IV), and α5(IV) chains cause the absence of the α3·α4·α5 network, which leads to progressive renal failure. In the present study, the molecular mechanism underlying the network defect was explored by further characterization of the chain organization and elucidation of the discriminatory interactions that govern network assembly. The existence of the two networks was further established by analysis of the hexameric complex of the noncollagenous (NC1) domains, and the α5 chain was shown to be linked to the α3 and α4 chains by interaction through their respective NC1 domains. The potential recognition function of the NC1 domains in network assembly was investigated by comparing the composition of native NC1 hexamers with hexamers that were dissociated and reconstituted in vitro and with hexamers assembled in vitro from purified α1-α5(IV) NC1 monomers. The results showed that NC1 monomers associate to form native-like hexamers characterized by two distinct populations, an α1·α2 and α3·α4·α5 heterohexamer. These findings indicate that the NC1 monomers contain recognition sequences for selection of chains and protomers that are sufficient to encode the assembly of the α1·α2 and α3·α4·α5 networks of GBM. Moreover, hexamer formation from the α3, α4, and α5 NC1 monomers required co-assembly of all three monomers, suggesting that mutations in the NC1 domain in Alport syndrome may disrupt the assembly of the α3·α4·α5 network by interfering with the assembly of the α3·α4·α5 NC1 hexamer.

Mechanism of Chain Selection in the Assembly of Collagen IV A PROMINENT ROLE FOR THE α2 CHAIN

Collagens comprise a large superfamily of extracellular matrix proteins that play diverse roles in tissue function. The mechanism by which newly synthesized collagen chains recognize each other and assemble into specific triple-helical molecules is a fundamental question that remains unanswered. Emerging evidence suggests a role for the non-collagenous domain (NC1) located at the C-terminal end of each chain. In this study, we have investigated the molecular mechanism underlying chain selection in the assembly of collagen IV. Using surface plasmon resonance, we have determined the kinetics of interaction and assembly of the α1(IV) and α2(IV) NC1 domains. We show that the differential affinity of α2(IV) NC1 domain for dimer formation underlies the driving force in the mechanism of chain discrimination. Given its characteristic domain recognition and affinity for the α1(IV) NC1 domain, we conclude that the α2(IV) chain plays a regulatory role in directing chain composition in the assembly of (α1)2α2 triple-helical molecule. Detailed crystal structure analysis of the [(α1)2α2]2 NC1 hexamer and sequence alignments of the NC1 domains of all six α-chains from mammalian species revealed the residues involved in the molecular recognition of NC1 domains. We further identified a hypervariable region of 15 residues and a β-hairpin structural motif of 13 residues as two prominent regions that mediate chain selection in the assembly of collagen IV. To our knowledge, this report is the first to combine kinetics and structural data to describe molecular basis for chain selection in the assembly of a collagen molecule.

ROLE OF CHOLESTEROL IN THE MODULATION OF INTERDIGITATION IN PHOSPHATIDYLETHANOLS

Phosphatidylethanol (Peth) is formed in biological membranes when ethanol replaces water in the transphosphatidylation reaction catalyzed by phospholipase D. This charged lipid accumulates in the presence of ethanol, and it has unusual properties that can influence membrane structure and function. We have previously shown that dimyristoylphosphatidylethanol (DMPeth) and dipalmitoylphosphatidylethanol (DPPeth) form the interdigitated gel phase in the presence of Tris-HCl [O.P. Bondar, E.S. Rowe, Biophys. J., 71 (1996) 1440-1449]. In the present investigation, differential scanning calorimetry (DSC) and fluorescence have been used to investigate the effect of cholesterol on the phase behavior of DPPeth and DMPeth. Our results show that cholesterol prevents the formation of the interdigitated phase in the presence of Tris-HCl, and that ethanol counters this influence and restores the ability of these lipids to interdigitate. Pyrene-PC fluorescence probe was used in this investigation and gave results that were in agreement with the conclusions based on the DSC study.

Effects of farnesol on the thermotropic behavior of dimyristoylphosphatidylcholine

Differential scanning calorimetry (DSC) and DPH fluorescence anisotropy have been used to investigate the effects of trans-trans farnesol on the physical properties of model membranes and extracted cell lipids. Farnesol was shown to have a significant effect on the gel to liquid-crystal phase transition temperature, the enthalpy of the transition and the transition co-operativity for extruded vesicles of dimyristoylphosphatidylcholine (DMPC). The phase transition of DMPC vesicles was eliminated at 25 mol% farnesol. Farnesol decreased the fluorescence anisotropy of the lipids extracted from human leukemia line CEM-C1 cells.