Mutant human lysozymes~Ile56Thr & Asp67His! have been reported to form amyloid deposits in the viscera. From the standpoint of understanding the mechanism of amyloid formation, we searched for conditions of amyloid formation in vitro using hen egg lysozyme, which has been extensively studied from a physicochemical standpoint. It was found that the circular dichroism spectra in the far-ultraviolet region of the hen egg lysozyme changed to those characteristic of a b-structure from the native a-helix rich spectrum in 90% ethanol solution. When the concentration of protein was increased to 10 mg0mL, the protein solution formed a gel in the presence of 90% ethanol, and precipitated on further addition of 10 mM NaCl. The precipitates were examined by electron microscopy, their ability to bind Congo red, and X-ray diffraction to determine whether amyloid fibrils were formed in the precipitates. Electron micrographs displayed unbranched protofilament with a diameter of ;70 Å. The peak point of the difference spectrum for the Congo red binding assay was 541 nm, which is characteristic of amyloid fibrils. The X-ray diffraction pattern showed a sharp and intense diffraction ring at 4.7 Å, a reflection that arises from the interstrand spacing in b-sheets. These results indicate that the precipitates of hen egg lysozyme are amyloid protofilament, and that the amyloid protofilament formation of hen egg lysozyme closely follows upon the destruction of the helical and tertiary structures.

The gradual removal of the denaturing reagent guanidine HCl (GdnHCl) using stepwise dialysis with the introduction of an oxidizing reagent and L-arginine resulted in the highly efficient refolding of various denatured single-chain Fv fragments (scFvs) from inclusion bodies expressed in Escherichia coli. In this study, the influence of the additives on the intermediates in scFv refolding was carefully analyzed on the basis of the stepwise dialysis, and it was revealed that the additive effect critically changes the pathway of scFv refolding. Circular dichroism and tryptophan fluorescence emission spectroscopies demonstrated that distinct secondary and tertiary structures were formed upon dialysis from 2 M GdnHCl to 1 M GdnHCl, and 4,4-dianilino-1,1-binaphthyl-5,5-disulfonic acid dipotassium salt binding analysis indicated that the addition of L-arginine to the stepwise dialysis system effectively stabilized the exposed hydrophobic area on the scFv. Quantification of the free thiol groups in the scFv by means of Ellman's assay revealed that there was a particular stage in which most of the free thiol groups were oxidized and that adding an oxidizing reagent (the oxidized form of glutathione, GSSG) at that stage was important for complete refolding of the scFv. The particular stage depended on the nature of the refolding solution, especially on whether L-arginine was present. Spontaneous folding at the 1 M GdnHCl stage resulted in a structure in which a free thiol group accessed to the proper one for correct disulfide linkage; however, the addition of Larginine resulted in the formation of a partially folded intermediate without disulfide linkages. Mass spectrometry experiments on alkylated scFv were carried out at each stage to determine the effects of L-arginine. The spectroscopic studies revealed two different pathways for scFv refolding in the stepwise dialysis system, pathways that depended on whether L-arginine was present. Controlled coupling of the effects of GSSG and L-arginine led to the complete refolding of scFv in the stepwise dialysis.The internal disulfide linkage in the immunoglobulin fold is of particular importance for most proteins in the immunoglobulin superfamily because this linkage has a critical influence on the stability of these proteins (1, 2). The linkage, which is highly conserved, connects two ␤-sheets in a sandwich structure (3-6). In the case of antibody molecules, Goto and Hamaguchi (1, 7) were the first to analyze intrachain disulfide bond formation in the constant fragments of the immunoglobulin light chain (C L ), 1 demonstrating the contribution of the disulfide linkage to the stabilization and folding of the C L domain. For the variable fragment in antibody molecules (Fv), which is composed of heavy and light chain domains (V H and V L , respectively), the immunoglobulin fold is known to be partially and irreversibly denatured to an aggregate under reducing conditions (2); furthermore, the instability of the Fv that is missing its disulfide linkages has been proved by natural a...

Biochemical and cellular properties of three immortalized Schwann cell lines expressing different levels of the myelin-associated glycoprotein (MAG) were compared . The S16 line generated by repetitive passaging was described previously and expresses a level of MAG comparable to that in adult sciatic nerve. The S42 line was generated independently by the same procedure, divides more slowly than the S16 line, and expresses an even higher level of MAG . The S1 6Y line arose spontaneously from a passage of the S16 cells, divides much more rapidly, and does not express MAG . The levels of MAG expression in the three lines are inversely related to their rates of proliferation, and MAG mRNA levels parallel the amounts of MAG . The S16 and S42 lines consist mainly of flat cells at low density and develop many processes at high density, whereas most of the S16Y cells are spindle-shaped, resembling primary Schwann cells in appearance . Surface immunostaining with the 04 antibody was positive for the S16 and S42 cells and negative for the S16Y cells, but all three lines were negative for surface staining with the 01 antibody . The overall protein compositions of the three lines are very similar, but the S16 and S42 cells express larger amounts of several glycoproteins than the S16Y cells, including the adhesion proteins, neural cell adhesion molecule, L1, and laminin . S16 and S42 cells (but not S1 6Y cells) also express P o glycoprotein, galactocerebroside, and sulfatide, but, unlike MAG, these other myelin-related components were present at much lower levels than in adult nerve . Myelin basic protein and proteolipid protein were not detected in any of the lines, although all three lines contained proteolipid protein mRNA . 2',3'-Cyclic nucleotide 3'-phosphodiesterase and glial fibrillary acidic protein were present in all three lines . Conditions have not yet been found in which any of the lines will myelinate dorsal root ganglion neurons in vitro, but the S16 and S42 cells differ from the S1 6Y cells by clustering around neurons after 1 week in coculture . In many respects, the S16 and S42 cells biochemically resemble Schwann cells at an early stage in their preparation to myelinate and should be useful for investigating the cell biology of MAG and other myelin-related components .

A point mutation in the gene for proteolipid protein (PLP) has been suggested to account for the dysmyelination seen in the jimpy mouse mutant. Despite the absence of PLP, the major integral membrane protein of central nervous system (CNS) myelin, this study shows that there are many scattered myelinated fibers present in the spinal cord of this murine mutant which are immunocytochemically positive for myelin basic protein (MBP), yet negative for PLP. This lack of PLP results in an abnormal compaction of the extracellular leaflets of the myelin sheath and the formation of an abnormal intraperiod line. These results are similar to those seen in another X-linked myelin mutant, the myelin-deficient rat (Duncan et al.: Proc. Natl. Acad. Sci. U.S.A., 84:6287-6291, 1987), and show that a multilamellar membrane can be formed in the absence of its major integral membrane protein.