1H and 15N NMR study of human lysozyme.

The 15N signal assignment of human lysozyme was carried out by using 1H-1H and 1H-15N two dimensional experiments. To solve the severe overlap problem of the NH signals, uniform labeling of the protein with 15N was introduced. The uniformly 15N labeled protein was prepared using a high-expression system of Saccharomyces cerevisiae. From the analyses of 1H and 15N NMR spectra, all of the backbone 15N signals of the molecule were assigned to each specific residue in the amino acid sequence. Recently published proton signal assignments [Redfield & Dobson (1990) Biochemistry, 29, 7201-7214] were confirmed by these complementary data. In addition, assignments were extended to side chain 15NH2 groups of asparagine and glutamine. Elements of secondary structure were deduced from the pattern of sequential and medium-range NOE connectivities. Two beta-sheets and four alpha-helices could be identified in the protein, which were in good agreement with those determined by X-ray crystallography. The interaction between human lysozyme and its inhibitor N-acetyl-chitotriose was investigated by 15N-1H HMQC spectra. Most of the 15N-NH cross-peaks in the spectra were separated well enough to be followed during the titration experiment. Residues whose NH proton signals decrease in intensity upon complex formation, are located mainly around subsites B, C, and D. Local conformational changes were observed around the fourth helix adjacent to the cleft of human lysozyme.     

Crystal structures of the apo- and holomutant human lysozymes with an introduced Ca2+ binding site

The three-dimensional structures of apo- and holomutant human lysozymes (D86/92 lysozyme), in which a calcium binding site was designed and created for enhancing molecular stability by replacing both Gln86 and Ala92 with aspartic acids, were refined at 1.8-A resolution by x-ray crystallography. The overall structures and crystallographic thermal factors of all three proteins, the apo-, holo-D86/92, and the wild-type human lysozymes, were essentially identical; these results showed that the introduction of the calcium binding site did not affect either the overall structure or molecular rigidity of the proteins. However, structure analyses of the apo-D86/92 lysozyme revealed that the mutations affected the side chain conformation of residue 86 and hydrogen networks between the protein and the internal solvent molecules. In the structure of the holo-D86/92 lysozyme, seven oxygen ligands formed a slightly distorted pentagonal bipyramid around the calcium ion, indicating that the coordination around the calcium ion was quite similar to that in baboon alpha-lactalbumin. The pentagonal bipyramid coordination could be one of the most widely found and appropriate calcium binding schemes in proteins.     

Alteration of N-terminal residues of mature human lysozyme affects its secretion in yeast and translocation into canine microsomal vesicles

Signal sequences play a central role in the initial membrane translocation of secretory proteins. Their functions depend on factors such as hydrophobicity and conformation of the signal sequences themselves. However, some characteristics of mature proteins, especially those of the N-terminal region, might also affect the function of the signal sequences. To examine this possibility, several mutants of human lysozyme modified in the N-terminal region of the mature protein were constructed, and their secretion in yeast as well as in vitro translocation into canine pancreatic microsomes were analyzed using an idealized signal sequence L8 (MR(L)8PLAALG). Our results show the following. (1) Change in the charge at the N-terminal residue of the mature protein does not affect secretion drastically. (2) Substitution of a proline residue at the N terminus prevents cleavage of the signal sequence, although translocation itself is not impaired. (3) Excessive positive charges in the N-terminal region delay translocation of the precursor protein across the membrane. (4) Polar and negatively charged residues introduced into the N-terminal region affect the secretion of the mature protein by preventing its correct folding.     

A novel cell surface antigen involved in thymocyte and thymic epithelial cell adhesion.

A murine mAb, 7D3, was produced by fusion of spleen cells obtained from mice immunized with a rat thymic epithelial cell line, Tu-D3 and NS/1 myeloma cells. 7D3 antibody reacted with approximately 95% thymocytes, 17% spleen cells, less than 9% of mesenteric lymph node cells and 32% of bone marrow cells of rat origin. 7D3 also reacted with two rat thymic epithelial cell lines but not with a rat fibroblastic cell line. Immunochemical analysis demonstrated that 7D3 antibody recognized a single polypeptide with molecular weight of 80,000 in FTE cells and 80,000 to 96,000 in thymocytes. 7D3 antibody strongly inhibited the thymocyte binding to thymic epithelial cells. In addition, 7D3 antibody inhibited TPA-induced thymocyte aggregation. 7D3 negative rat thymic lymphoma cells bound to 7D3 positive thymic epithelial cells and this binding was inhibited by 7D3 antibody, indicating that a part of thymocyte-thymic epithelial cell binding was mediated by the interaction of 7D3 Ag and undefined ligand to 7D3.     

Collagen gel contraction by fibroblasts requires cellular fibronectin but not plasma fibronectin

Fibroblasts embedded in three-dimensional lattices of collagen fibrils have been known to require serum constituents to induce a cell-mediated contraction of collagen gels. The gel contraction was studied with human skin fibroblasts cultured in the presence of fetal bovine serum (FBS). Removal of bovine serum fibronectin (sFN) from FBS did not affect the extent of gel contraction. Gel contraction occurred in serum-free defined media. Therefore, it is concluded that sFN is not required for gel contraction. That cellular FN (cFN) synthesized and secreted by fibroblasts plays a crucial role in gel contraction was suggested by the following experiments: (1) We obtained monoclonal antibodies (mAb A3A5) against fibroblast surface antigens, which suppressed the fibroblast-mediated gel contraction. Immunoblot analyses showed that mAb A3A5 recognizes cFN secreted by human fibroblasts and human plasma FN (pFN), but not bovine sFN in FBS used for culture. (2) Addition of rabbit antisera, which recognize human cFN, to a serum-free gel culture inhibited contraction. Uninvolvement of human pFN in gel contraction was further confirmed by the fact that neither pretreatment of fibroblasts with excess amounts of human pFN nor the presence of excess amounts of human pFN in gels affected the extent of gel contraction. This study seems to be the first demonstration of functional difference between cFN and pFN (or sFN) and proposes a novel mode of binding of fibroblasts with collagen fibrils via cFN during cell-mediated collagen morphogenesis.     

Effects of Hypoxia on the Activity of the Dopaminergic Neuron System in the Rat Striatum as Studied by In Vivo Brain Microdialysis

The purpose of the present study is to clarify the effects of hypoxia on the activity of the dopaminergic neurons in the brain and its mechanism of action. For this purpose, the effects of hypoxia on the extracellular levels of 3,4-dihy-droxyphenylethylamine (dopamine) were examined in the rat Striatum using in vivo brain microdialysis in the presence or absence of pretreatment with either tetrodotoxin (a blocker of voltage-dependent sodium channels) or nomifensine (a blocker of dopamine reuptake). Exposure to various degrees of hypoxia (15, 10, and 8% O₂ in N₂) increased dopamine levels in striatal dialysates to 200, 400, and 1,100%, respectively, of the control value. On reoxygenation, dopamine levels in the dialysates rapidly returned to the control level. Reexposure to hypoxia increased the dopamine levels to the same extent as during the first exposure. After addition of tetrodotoxin (40 mUM) to the perfusion fluid or pretreatment with nomifensine (100 mg/kg, i.p.), exposure to hypoxia no longer increased the dopamine levels. These results suggest that although hypoxia induces an increase in the extracellular dopamine levels (hence, an apparent increase in the activity of the dopaminergic neurons), this increase is not the result of an increase in dopamine release itself, but rather the result of inhibition of the dopamine reuptake mechanism.     

Assignment of disulphide bonds in synthetic endothelin-1 isomers by fast atom bombardment mass spectrometry.

Endothelin-1 (ET-1), a 21-residue vasoconstrictor peptide possessing four cysteinyl residues at positions 1, 3, 11 and 15, was synthesized by random oxidation of a tetrahydro-ET-1. On reverse-phase high-performance liquid chromatography, crude product was shown to be a mixture of two disulphide isomers. A method was developed to determine the disulphide structure of the isomers. The method consisted of (a) limited digestion with chymotrypsin, (b) cleavage with cyanogen bromide and (c) manual Edman degradation. Through this procedure, each isomer afforded specific fragments containing a single disulphide bond, which were identified by fast atom bombardment mass spectrometry. Isomer 1, the minor component, afforded a fragment containing Cys 3 and Cys 15, and isomer 2, the major component, afforded fragments containing Cys 3 and Cys 11. Since little disulphide exchange was observed, it could be concluded clearly that the disulphide bond pairs in isomer 1 were Cys 1-Cys 11 and Cys 3-Cys 15, while those in isomer 2 were Cys 1-Cys 15 and Cys 3-Cys 11 (the same as natural ET-1). The procedure was successfully applied to two synthetic analogues, [Gly18]-ET-1 and [Pro16]-ET-1.     

Increase in potential activities of protein phosphatases PP1 and PP2A in lymphoid tissues of autoimmune MRL/MpJ-lpr/lpr mice.

The differential assay conditions for protein phosphatases PP1, PP2A, and PP2C were extensively studied by using crude extracts from mouse lymphoid tissues as enzyme sources. Under these conditions, the protein phosphatase activities were measured in MRL/MpJ-lpr/lpr mice (MRL/lpr mice), autoimmune-prone mice, and MRL/MpJ(-)+/+ mice (MRL/+/+ mice) and C3H/HeJ mice as the controls. In MRL/lpr mice, significant alterations in protein phosphatase activities from those in the control mice were demonstrated. In spleen and liver from MRL/lpr mice, potential activities of PP1 and PP2A were distinctly elevated over those of the control mice. These elevations appeared to be due to accumulation of the abnormal lymphocytes that emerged in MRL/lpr mice. Although the PP1 activity in MRL/lpr lymph nodes was lower than those of normal spleen and thymus, it was greatly increased by Co(2+)-trypsin treatment so that the PP1 activity of MRL/lpr lymph nodes was the highest among those of all the tissues examined. In contrast, PP2C activity showed no remarkable alteration in the autoimmune disease model mice as compared with that in the control mice. These results demonstrated a specific elevation in potency of protein dephosphorylation in the tissues of MRL/lpr mice, suggesting a new explanation for the defect in signal transduction in this disease.