Lipids and proteins in multiple sclerosis white matter

Abstract— Quantitative analyses of white matter from four brains of patients with multiple sclerosis (MS) and four control brains were carried out for total and soluble proteins, individual lipid fractions, and their corresponding fatty acids. In three specimens from two of the MS brains there were reductions of cerebrosides and of the C_20:1 acid in the ethanolamine glycerophosphatide (EGP) fraction and a slight increase of tetraenes and trienes, while all other components were present in concentrations similar to those in the controls. In three other samples from two of the MS brains, galactolipids were deficient to a greater extent than cholesterol, EGP or CGP (choline glycerophosphatide), while proteins were within the control range. In samples where thinning of myelin was observed in Luxol-blue stained sections, there were proportional decreases of all components. The percentage of C_20:1 acid in the EGP fraction was reduced in two of three myelin preparations from corresponding samples of MS white matter, and that of C_24:1 acid in the cerebroside fraction was reduced in all three MS myelin preparations when compared with the two controls. The data suggest that inadequacy of the fatty acid elongation process together with deficits of cerebrosides represent one of the early biochemical lesions in the white matter of the MS brain.     

The isolation of glycoproteins from bovine achilles tendon and their interaction with collagen

Two glycoprotein fractions, A and B, were isolated from bovine achilles tendon. Glycoprotein A was prepared from a 0.2m-sodium chloride extract and glycoprotein B was isolated from a 3m-magnesium chloride extract. They were free from serum proteins. Glycoprotein A was essentially free of collagen, but glycoprotein B contained about 8% collagen. Both glycoproteins gave several bands on isoelectric focusing. This technique was also used to demonstrate that both glycoprotein fractions interacted strongly with acid-soluble calf skin tropocollagen. It is concluded that these fractions are true components of tendon, and that they may have some function in the macromolecular organization of the tissue.     

Inhibition and promotion of cholesterol crystallization by protein fractions from normal human gallbladder bile

Pooled, normal human gallbladder biles were initially separated on a molecular sieving chromatography column to remove soluble mucin glycoproteins as well as high molecular weight proteins (greater than 200,000). The remaining lower molecular weight proteins and other bile components were then examined by lectin affinity chromatography with four different types of lectin. The separated bound fractions were compared for inhibiting and promoting activities with a newly devised sensitive cholesterol crystal growth assay and for differences in electrophoretic patterns on SDS-gels. Protein factors (presumably glycoproteins) were found to have both inhibiting and promoting activities, even in the absence of cholesterol gallstone disease. The promoting effect was indicated by shortened crystal detection times and increases in crystal growth rate; whereas the inhibiting effect was indicated by decreases in crystal growth rate and reductions in the final crystal concentration as determined by the growth assay. Affinity chromatography mitigated the major problems of removing both lipids and pigment from the glycoproteins. In addition, partial purification of bound fractions with potent cholesterol crystal nucleation-altering activity can be obtained by this technique.     

茶树叶糖蛋白分离及鉴定的初步研究

茶叶是一种有益健康的保健食品,保健功能在于它有多种化学活性成分。近年来研究表明,茶叶糖蛋白可能是茶叶的保健因子之一。从茶树叶中提取了总蛋白、45%~70%硫酸铵分级总蛋白、分级蛋白Sephadex G-100凝胶层析分离出粗糖蛋白,根据糖蛋白糖链及蛋白的对照染色鉴定出多种茶树叶糖蛋白,并从SDS-胶上快速纯化了三种茶叶糖蛋白。ConA-Sepharose 4B亲和层析从茶树叶总蛋白中分离出16种天然活性糖蛋白。     

Proteomic and functional alterations in brain mitochondria from Tg2576 mice occur before amyloid plaque deposition

Synaptic dysfunction is an early event in Alzheimer's disease patients and has also been detected in transgenic mouse models. In the present study, we analyzed proteomic changes in synaptosomal fractions from Tg2576 mice that overexpress mutant human amyloid precursor protein (K670N, M671L) and from their nontransgenic littermates. Cortical and hippocampal tissue was microdissected at the onset of cognitive impairment, but before deposition of amyloid plaques. Crude synaptosomal fractions were prepared by differential centrifugation, proteins were separated by 2-D DIGE and identified by MS/MS. Significant alterations were detected in mitochondrial heat shock protein 70 pointing to a mitochondrial stress response. Subsequently, synaptosomal versus nonsynaptic mitochondria were purified from Tg2576 mice brains by density gradient centrifugation. Mitochondrial proteins were separated by IEF or Blue-native gel electrophoresis in the first dimension and SDS-PAGE in the second dimension. Numerous changes in the protein subunit composition of the respiratory chain complexes I and III were identified. Levels of corresponding mRNAs remain unchanged as shown by Affymetrix oligonucleotide array analysis. Functional examination revealed impaired state 3 respiration and uncoupled respiration in brain mitochondria from young Tg2576 mice. By immunoblotting, amyloid-beta oligomers were detected in synaptosomal fractions from Tg2576 mice and reduced glucose metabolism was observed in Tg2576 mice brains by [14C]-2-deoxyglucose infusion. Taken together, we demonstrate alterations in the mitochondrial proteome and function that occur in Tg2576 mice brains before amyloid plaque deposition suggesting that mitochondria are early targets of amyloid-beta aggregates.     

Frozen tissue can provide reproducible proteomic results of subcellular fractionation

Differential detergent fractionation (DDF) is frequently used to partition fresh cells and tissues into distinct compartments. We have tested whether DDF can reproducibly extract and fractionate cellular protein components from frozen tissues. Frozen kidneys were sequentially extracted with three different buffer systems. Analysis of the three fractions with liquid chromatography–tandem mass spectrometry (LC–MS/MS) identified 1693 proteins, some of which were common to all fractions and others of which were unique to specific fractions. Normalized spectral index (SI_N) values obtained from these data were compared to evaluate both the reproducibility of the method and the efficiency of enrichment. SI_N values between replicate fractions demonstrated a high correlation, confirming the reproducibility of the method. Correlation coefficients across the three fractions were significantly lower than those for the replicates, supporting the capability of DDF to differentially fractionate proteins into separate compartments. Subcellular annotation of the proteins identified in each fraction demonstrated a significant enrichment of cytoplasmic, cell membrane, and nuclear proteins in the three respective buffer system fractions. We conclude that DDF can be applied to frozen tissue to generate reproducible proteome coverage discriminating subcellular compartments. This demonstrates the feasibility of analyzing cellular compartment-specific proteins in archived tissue samples with the simple DDF method.     

Molecular characteristics of the major scrapie prion protein

A major protein was identified that purifies with the scrapie agent extracted from infected hamster brains. The protein, designated PrP 27-30, was differentiated from other proteins in purified fractions containing the scrapie agent by its microheterogeneity (Mr 27000-30000) and its unusual resistance to protease digestion. PrP 27-30 was found in all fractions enriched for scrapie prions by discontinuous sucrose gradient sedimentation or sodium dodecyl sarcosinate-agarose gel electrophoresis. It is unlikely that PrP 27-30 is a pathologic product because it was found in fractions isolated from the brains of hamsters sacrificed prior to the appearance of histopathology. If PrP 27-30 is present in normal brain, its concentration must be 100-fold lower than that found in equivalent fractions from scrapie-infected hamsters. Three protease-resistant proteins similar to PrP 27-30 were found in fractions obtained by discontinuous sucrose gradient sedimentation of scrapie-infected mouse brain. These proteins were not evident in corresponding fractions prepared from normal mouse brain. One-dimensional peptide maps comparing PrP 27-30 and normal hamster brain proteins of similar molecular weight demonstrated that PrP 27-30 has a primary structure which is distinct from these normal proteins. Heating substantially purified scrapie fractions to 100 degrees C in sodium dodecyl sulfate inactivated the prion and rendered PrP 27-30 susceptible to protease digestion. Though the scrapie agent appears to be hydrophobic, PrP 27-30 remained in the aqueous phase after extraction with organic solvents, indicating that it is probably not a proteolipid. PrP 27-30 is the first structural component of the scrapie prion to be identified.     

Extractability of glycoproteins and mucopolysaccharides of brain

Very little is known about the localization and functions of the glycoproteins and mucopolysaccharides of nervous tissue. There have been two major approaches to the study of these substances in brain. The first involves the isolation of glycopeptides and mucopolysaccharides after digestion of the lipid-free protein residue from whole brain with proteolytic enzymes (Margolis , 1967; Di Benedetta et al., 1969; Margolis and Margolis , 1970; Katzman , 1972). This approach has the advantage that sufficient tissue is used to permit analysis of the structure and metabolism of the carbohydrate components of these macromolecules. However, any differentiation of the various glycoproteins and mucopolysaccharides based on such features as their anatomical location, association with proteins, lipids or other membrane components, and the properties conferred by their non-carbohydrate portion, is unavoidably lost as a consequence of the procedures used for their isolation. On the other hand, several laboratories have attempted to study the glycoproteins and mucopolysaccharides of nervous tissue by treating brain (or subcellular fractions) with various detergents, and then examining the extracts for the pattern of separation obtained by polyacrylamide gel electrophoresis in terms of carbohydrate staining reactions or the incorporation of labelled precursors (Bosmann , Case and Shea , 1970; Duiton and Barondes , 1970; Quarles and Brady , 1971; Waehneldt , Morgan and Gombos , 1971). This approach has the advantages of relatively high sensitivity and the ability to study intact glycoproteins rather than glycopeptides produced by proteolytic enzyme digestion. However, it is presently impossible to identify any of the numerous and often poorly resolved bands thus obtained with glycoproteins or mucopolysaccharides of known structure and chemical composition, or in many cases even to identify the various complex carbohydrates as being glycoproteins, glycolipids or acid mucopolysaccharides. In an attempt to obtain some indication of the degree of anatomical heterogeneity of these compounds in nervous tissue, we have sequentially treated whole rat brain with several solvents to obtain intact glycoproteins and mucopolysaccharides. After removal of lipids and digestion with pronase, the composition of the glycopeptides and mucopolysaccharides has been analyzed.     

Integrated mass spectrometry-based analysis of plasma glycoproteins and their glycan modifications

We present a protocol for the identification of glycosylated proteins in plasma followed by elucidation of their individual glycan compositions. The study of glycoproteins by mass spectrometry is usually based on cleavage of glycans followed by separate analysis of glycans and deglycosylated proteins, which limits the ability to derive glycan compositions for individual glycoproteins. The methodology described here consists of 2D HPLC fractionation of intact proteins and liquid chromatography-multistage tandem mass spectrometry (LC-MS/MS(n)) analysis of digested protein fractions. Protein samples are separated by 1D anion-exchange chromatography (AEX) with an eight-step salt elution. Protein fractions from each of the eight AEX elution steps are transferred onto the 2D reversed-phase column to further separate proteins. A digital ion trap mass spectrometer with a wide mass range is then used for LC-MS/MS(n) analysis of intact glycopeptides from the 2D HPLC fractions. Both peptide and oligosaccharide compositions are revealed by analysis of the ion fragmentation patterns of glycopeptides with an intact glycopeptide analysis pipeline.     

Fractionation of desmosomes and comparison of the polypeptide composition of desmosomes prepared from two bovine epithelial tissues

Desmosomes isolated from bovine tongue mucosa or muzzle epidermis appeared identical by ultrastructural analyses but had some differences in their polypeptide compositions as determined by SDS-PAGE. These preparations were extracted in 9 M urea, 10 mM Tris-HCl (pH 9), and 25 mM B-mercaptoethanol and then centrifuged at 240,000g for 30 min. The urea-soluble and insoluble fractions were analyzed by SDS-PAGE. The urea soluble fractions of both tongue and muzzle desmosomes were enriched in polypeptides of 240, 210, 81, and 75 kDa and also polypeptides (40 to 70 kDa) that were keratin-like, as determined by immunoblotting analyses with keratin antisera. The urea insoluble fraction of tongue desmosomes contained glycoproteins of 165, 160, 140, 110, and 100 kDa, while this fraction from muzzle contained glycoproteins of 165, 115, and 105 kDa. Ultrastructural examinations of insoluble pellets obtained from urea extracted tongue and muzzle desmosomes showed that most of the components at the cytoplasmic faces of the desmosomes were removed, while the membrane regions of the desmosomes resisted the treatment. The urea soluble proteins were dialyzed against 10 mM Tris-HCl (pH 7.6), and the resulting preparation was pelleted by centrifugation and examined by electron microscopy. Ultrastructural examination of this material revealed that it had assembled into a fibrillar meshwork, similar to the fibrillar region adjacent to the submembranous plaque of isolated desmosomes. Thus, treatment of isolated desmosomes with 9 M urea allowed the fractionation of membrane-associated desmosomal proteins from cytoplasmic desmosomal proteins. A comparison of these fractions from tongue and muzzle indicated that the polypeptide compositions of the desmosomes varied between tissues, especially with respect to the fractions enriched in either glycoproteins or keratin.     

Elevated levels of a glycoprotein antigen (P-80) in gray and white matter of brain from victims of multiple sclerosis

The levels of a glycoprotein reactive with monoclonal antibody (MAb) 44D10 in white and gray matter from brains of victims of several neurological diseases, including Multiple Sclerosis, Alzheimer's, Parkinson's and Huntington's diseases, were compared to that of normal individuals. The concentration of antigen reactive with MAb 44D10 was elevated in both gray and white matter of all MS brains examined, but not in brains with other neurological diseases. The increase in the concentration of antigen varied amongst the MS brains, such that the levels of antigen were only slightly increased in 2 of the 6 MS brains whereas 2 to 4 fold higher levels were found in the other 4 brains. Increased levels of antigen were detected in gray matter of MS brains, whereas this antigen was either not detected or present in very low levels in gray matter homogenates prepared from agematched normal brains. MAb Leu 1, which reacts with T lymphocytes, was not absorbed by normal and MS brain tissue suggesting the increase in antigen reactive with MAb 44D10 in MS brain homogenates was not associated with non-specific infiltration by T lymphocytes. Comparison of the purified antigen from MS gray matter and normal white matter by gel electrophoresis demonstrated that MAb 44D10 was reacting with a similar protein in both tissues with an apparent molecular weight of 80K. We have named this molecule P-80 glycoprotein.     

Evidence of Nitrosative Damage in the Brain White Matter of Patients with Multiple Sclerosis

Nitric oxide (NO) has been implicated in the pathophysiology of both experimental autoimmune encephalomyelitis and multiple sclerosis (MS). NO-mediated protein damage in MS appears to be confined to large plaques where 3-nitrotyrosine has been detected. To determine whether nitrosative damage takes place beyond visible MS plaques, the occurrence of various NO-triggered protein modifications in normal-appearing white matter (NAWM) of eight MS brains was assessed and compared to that in white matter (WM) of four control brains. As determined by amino acid analysis and western blotting, no evidence of tyrosine nitration was found in the MS samples studied, suggesting that they did not contain appreciable amounts of plaque-derived material. The amino acid composition of total myelin proteins and proteolipid protein (PLP) was also unaltered in the diseased tissue, as was the fatty acid composition of PLP. In addition, we detected no changes in the number of protein free thiols suggesting that oxidation do not occur to any appreciable extent. However, the levels of nitrite in MS-NAWM were higher than those in control WM, while in the MS-gray matter (GM) the concentration of this ion was unaltered. Furthermore, five of the MS samples analyzed, and the same as those with high levels of glial fibrilary acidic protein, showed increased amounts of protein nitrosothiols as determined by the biotin switch method. S-nitrosation of GM proteins was again normal. There was no indication of N-nitrosation of tryptophan and N-terminal amino groups in both control and MS tissue. Overall, the data suggests that WM, but not GM, from MS brains is subjected to considerable nitrosative stress. This is the first report to present direct evidence of increased protein S-nitrosation and nitrite content in the brain parenchyma of MS patients.     

Characterization of Postmortem Human Brain Proteins by Two-Dimensional Gel Electrophoresis

Abstract: The proteins of membrane and cytosol fractions from frozen human postmortem brain were analyzed by two-dimensional gel electrophoresis (isoelectric range: 5.1–6.0) and both Coomassie-blue and ammoniacal silver staining. Cytosol preparations were analyzed from six different postmortem brains from patients with various neurologic diagnoses and immediate causes of death. Intervals between death and brain freezing (−70^oC) ranged from 2 to 20 h. The vast majority of proteins detected in these cytosol fractions had identical molecular weights and isoelectric points in each of six human brains examined. However, in some tissue samples tubulin was either quantitatively decreased or undetectable. The possibility that this partial or complete depletion of tubulin was related to postmortem interval and/or brain freezing was studied using rat forebrain tissue. Rat brain incubated at room temperature for up to 24 h did not reproduce the changes seen in the region of human cytosol tubulin. However, other changes seen in the two-dimensional electrophoretic pattern of rat cytosol proteins did relate to postmortem interval, brain freezing, or both. Rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum were prepared from three human brains, with highly reproducible two-dimensional patterns. Protein analysis of these membrane fractions revealed that human RER contained significant amounts of tubulin, in contrast to rat RER which contained no detectable tubulin. This discrepancy was elucidated by allowing rat brains to remain at room temperature for 24 h before freezing; gels of rat RER prepared from this tissue showed that tubulin subunits were present.     

Cytoplasmic casein kinase 2 and its substrate proteins in the brain in Alzheimer's disease]

The absence of casein kinase 2 on blots of temporal cortex extracts from Alzheimer's disease patients (ADP) was shown using antiserum to casein kinase 2. Casein kinase 2 activity towards endogenous substrates and casein is 2-5 times less in ADP brain in comparison to normal controls. The fractions of heparin-binding proteins, containing protein substrates for phosphorylation, were isolated from temporal cortex of ADP and normal controls. The total amount of heparin-binding proteins from ADP brains is less than from control brains, and the polypeptide composition of these fractions is much more poop.     

20-Hydroxyecdysone increases the metabolic labeling of extracellular glycoproteins of Drosophila S3 cells

20-Hydroxyecdysone (20-HOE) induces evagination of imaginal discs of Drosophila and aggregation in certain Drosophila cell lines. During both evagination and aggregation extensive changes in cell surface proteins occur. Immunological cross-reactivity has been demonstrated between certain hormone-dependent cell surface proteins in discs and cell lines, although apparently identical proteins are more easily solubilized in cell lines than in imaginal discs. These and other observations suggest that in imaginal discs some of these proteins might be basal lamina or extracellular matrix components. Therefore we have investigated the possbility that in the hormone-responsive cell line S3, certain proteins metabolically labeled in a hormone-dependent fashion might be released into the medium. Our results demonstrate for the first time that Drosophila tissue culture cells produce an array of extracellular glycoproteins, and that the metabolic labeling of several of these glycoproteins is increased substantially by 20-HOE. The presence of these labeled glycoproteins in the medium is decreased reversibly by the ionophore monensin, suggesting that this is a Golgi-mediated process. Several hormone-dependent extracellular glycoproteins are immunoprecipitated by an antiserum raised against imaginal disc cell membranes. During hormone-dependent reaggregation of S3 cells, the appearance of several hormone-dependent glycoproteins in the medium coincides with the onset and continuation of reaggregation. We suggest that these glycoproteins may function in hormone-induced cell-cell interactions during S3 cell aggregation. We also hypothesize that these hormone-dependent glycoproteins may function during in vivo morphogenesis as basal lamina and/or extracellular matrix components.     

Analysis of lectin-bound glycoproteins in snake venom from the Elapidae and Viperidae families

This paper describes an efficient method of studying the glycoproteins found in snake venom. The glycosylation profiles of the Elapidae and Viperidae snake families were analyzed using FITC-labeled lectin glycoconjugates. The Con A-agarose affinity enrichment technique was used to fractionate glycoproteins from the N. naja kaouthia venom. The results revealed a large number of Con A binding glycoproteins, most of which have moderate to high molecular weights. To identify the proteins, the isolated glycoprotein fractions were subjected to two-dimensional electrophoresis and MALDI-TOF MS. Protein sequences were compared with published protein databases to determine for their biological functions.     

Identification and verification of novel rodent postsynaptic density proteins

The postsynaptic density (PSD) is a cellular structure specialized in receiving and transducing synaptic information. Here we describe the identification of 452 proteins isolated from biochemically purified PSD fractions of rat and mouse brains using nanoflow HPLC coupled to electrospray tandem mass spectrometry (LC-MS/MS). Fluorescence microscopy and Western blotting were used to verify that many of the novel proteins identified exhibit subcellular distributions consistent with those of PSD-localized proteins. In addition to identifying most previously described PSD components, we also detected proteins involved in signaling to the nucleus as well as regulators of ADP-ribosylation factor signaling, ubiquitination, RNA trafficking, and protein translation. These results suggest new mechanisms by which the PSD helps regulate synaptic strength and transmission.     

Preparation and properties of nexuses and lipid-enriched vesicles from mouse liver plasma membranes

Extraction of mouse liver plasma membranes with 4% (w/v) N-laurylsarcosinate-tris buffer, pH7.8, solubilized 80-90% of the protein and 60% of the 5'-nucleotidase activity. The membrane residue remaining after extraction was resolved on sucrose gradients into two fractions: a vesicular membrane fraction and a fraction characterized by the presence of large numbers of nexuses in an amorphous background. The vesicular fraction had a phospholipid/protein weight ratio of 7:1, it contained most of the plasma-membrane glycolipids, and polyacrylamide-gel electrophoresis indicated the presence of only five to eight proteins, including two or three glycoproteins. The 5'-nucleotidase and leucine naphthylamidase specific activities were 23- and 6-fold higher respectively than in the plasma membranes. Electron microscopy of thin sections and negatively stained preparations indicated that the nexuses present in the second fraction closely resembled gap junctions present in tissue sections and isolated plasma membranes. The nexus fraction contained a distinctive protein pattern, and of the 20 proteins present about four were identified as glycoproteins by Schiff-periodate staining. Examination of the lipid composition of the fractions by t.l.c. showed that in the nexus fraction, phospholipids and glycolipids were present in small amounts compared with triglycerides and cholesterol. Amino sugar analyses confirmed the t.l.c. results and amino acid analysis showed the fractions to have characteristic protein compositions. A ;reconstituted' membranous fraction prepared by dialysis against MgCl(2) of membrane components soluble in N-laurylsarcosinate-tris buffers, pH7.8, lacked the trilaminar image characteristic of the two other membrane fractions isolated and was devoid of enzyme activities. The results indicate that proteins and glycoproteins play an important role in the structural maintenance of the nexuses isolated from the liver by the present procedure.     

Studies on bone matrix glycoproteins. Incorporation of [1-14C]glucosamine and plasma [14C]glycoprotein into rabbit cortical bone

The radioactively labelled constituents present in bone matrix were compared 12 days after injection of either [(14)C]glucosamine or plasma [(14)C]glycoprotein. Both precursors are utilized in the synthesis of organic matrix by bone tissue. Cortical bone from animals injected with [(14)C]glucosamine contains radioactivity derived from glucosamine and plasma glycoproteins and all glycoprotein fractions are labelled. Plasma [(14)C]glycoprotein labels the less acidic glycoproteins to a greater extent than the more acidic components. An antibody has been raised against the less-acidic-glycoprotein fraction of bone. The latter contains a glycoprotein of alpha-mobility that appears to be concentrated specifically in bone tissue and which is present also in plasma. This alpha-glycoprotein accounts for a large proportion of the components labelled and retained in bone matrix after [(14)C]glucosamine injection.