Neuroprotection by α-Lipoic Acid in Streptozotocin-Induced Diabetes

Glial cells provide structural and metabolic support for neurons, and these cells become reactive to any insult to the central nervous system. The streptozotocin (STZ) rat model was used to study glial reactivity and the prevention of gliosis by alpha-lipoic acid (alpha-LA) administration. The expression of glial fibrillary acidic protein (GFAP), S100B protein, and neuron specific enolase (NSE) was determined as well as lipid peroxidation (LPO) and glutathione (GSH) levels in some brain tissues. Western blot analyses showed GFAP, S100B, and NSE levels significantly increased under STZ-induced diabetes in brain, and LPO level increased as well. Administration of alpha-LA reduced the expression both of glial and neuronal markers. In addition, alpha-LA significantly prevented the increase in LPO levels found in diabetic rats. GSH levels were increased by the administration of alpha-LA. This study suggests that alpha-LA prevents neural injury by inhibiting oxidative stress and suppressing reactive gliosis.     

A balanced expression of two chains of heterodimer protein, the human interleukin-12, improves high-level expression of the protein in CHO cells

Interleukin-12 (IL-12) comprises of p40 and p35 subunits that are encoded by genes on separate chromosomes and form p70 heterodimer, a bioactive protein, and free p40, an antagonist of IL-12. Balance expression of two subunits within cells would be the key for high-level of production of bioactive IL-12. Thinking about different expression efficiencies of two genes (p40 gene with higher efficiency), we selected two expression vectors with different efficiencies and inserted genes of p40 and p35 into them separately and co-transfected them into Chinese hamster ovary (CHO) cells. The high-level expression of IL-12 was obtained when p40 cDNA was inserted into pcDNA3 (lower expression vector) and p35 cDNA was inserted into pEE14 (higher expression vector), but using pEE14 for p40 cDNA and pcDNA3 for p35 cDNA, which was opposite to the optimal design, or pEE14 or pcDNA3 for both p35 cDNA and p40 cDNA did not obtain high-level of production of p70 heterodimer, the bioactive IL-12. We also observed that using two chemical reagents in combination, as a pressure selection method or amplification for the two vectors, markedly enhanced the IL-12 production, when compared with any one selection chemical. Our results indicated that the balance expressions of two chains of hetrodimer protein, such as p40 and p35 of IL-12, would be a better choice to obtain high-level of production of the proteins.     

Regulation of rat mammary gene expression by extracellular matrix components

In the mammary gland the induction and maintenance of differentiation are dependent on both lactogenic hormones and the extracellular matrix (ECM). Since mammary epithelial cells differentiate on a basement membrane in vivo we have examined the effects of basement membrane components on the expression of milk protein genes in primary rat mammary cultures. We examined the effects of a basement membrane gel derived from the Englebreth-Holm-Swarm tumor as well as its major component, laminin, on the expression of a group of milk protein genes. We demonstrate that the basement membrane gel induces alpha-casein and alpha-lactalbumin (alpha-LA) accumulation up to 160- and 70-fold, respectively, of that on tissue culture plastic. Laminin, a major component of the basement membrane, also caused significant induction of these same proteins. In order to determine whether these ECM effects occurred at a translational or post-translational level, pulse-chase experiments were performed. These experiments demonstrated that a laminin substratum selectively effects milk protein turnover and secretion. In order to demonstrate whether ECM effects occurred at the level of steady state accumulation of mRNA we performed dot blot and Northern analyses using cloned cDNA probes for alpha-, beta-, and gamma-caseins and alpha-LA. These studies demonstrated that ECM components induced alpha- and beta-caseins up to 10-fold, and alpha-LA up to 3-fold, with no significant effect on gamma-casein. These results demonstrate that milk protein genes are not coordinately regulated by ECM components. Furthermore, since the amount of induction of milk proteins exceeds the amount of induction of mRNAs for these proteins, we conclude that in our system a major effect of ECM components is at the translational and/or post-translational levels. Based on these findings we propose a model in which basement membrane components effect mammary gene expression at multiple levels.     

Stability of the molten globule state of a domain-exchanged chimeric protein between human and bovine alpha-lactalbumins

A domain-exchanged chimeric alpha-lactalbumin (alpha-LA), which consisted of the alpha-domain of human alpha-LA and the beta-domain of bovine alpha-LA, was constructed. Like native alpha-LA, the chimeric protein was in a molten globule state in the absence of Ca(2+) at neutral pH and low salt concentration. The stability of the molten globule state of the constructed chimeric protein was identical to that of the recombinant human protein and was higher than that of the recombinant bovine protein. The stability of the molten globule state of alpha-LA is defined by the stability of the alpha-domain.     

Use of silent mutations in cDNA encoding human glutathione transferase M2-2 for optimized expression in Escherichia coli.

Heterologous expression of human glutathione transferase M2-2 (GST M2-2) using Escherichia coli was improved 140-fold by mutating the cDNA expressing the enzyme. Expression of GST M2-2 from this cDNA clone, pKHXhGM2, generated approximately 190 mg protein per liter of bacterial culture, corresponding to approximately 12% of the total amount of soluble protein. The high-level-expressing cDNA was generated by oligonucleotide-directed mutagenesis introducing alternative silent mutations into the third nucleotide of codons 2, 4-7, and 10-14 in the 5' end of the cDNA coding region. The choice of alternative codons was restricted to those naturally occurring in highly biased genes in E. coli. Furthermore, the wild-type TAG stop codon at the 3' end was replaced with the two stop codons TAA and TGA in tandem to increase translation termination efficiency. The resulting partially randomized cDNA library was assayed for high-level expression using immunoscreening. Sequence similarities between the constructed high-level-expressing GST M2-2 cDNA and a similarly designed cDNA encoding the closely related human GST M1-1 suggest that the codons in the region immediately following the start codon are influential in achieving high-level expression. Pyrimidines seem to be more favorable than purines in the third position of codons in optimizing the expression of these enzymes in E. coli.     

Zinc binding in bovine alpha-lactalbumin: sequence homology may not be a predictor of subtle functional features

alpha-Lactalbumin (alpha-LA), a calcium-binding protein, also possesses zinc-binding sites comprising a single strong site and several weaker secondary sites. The only site found by X-ray crystallography (Ren et. al., J. Biol. Chem. 1993;268:19292) was Glu 49 of human alpha-LA, but zinc binding had never been measured in solution for human alpha-LA. This residue was genetically substituted by Ala in bovine alpha-LA and the metal-binding properties of the resulting desMetE49A protein were compared with those for native alpha-LA by fluorescence methods. Surprisingly, desMetE49A alpha-LA and the native bovine protein had similar affinities for both Zn(2+) and Ca(2+). Genetic substitution of other possible candidates for Zn(2+) chelating residues, which included Glu 25, did not alter the affinity of bovine alpha-LA to Zn2+; however, substitution of Glu 1 by Met resulted in the disappearance of strong Zn(2+) binding. A proposed site involves Glu 1, Glu 7, Asp 11, and Asp 37, which would participate in strong Zn(2+) binding based on their propinquity to Glu 1. Human alpha-LA, which has a Lys at position 1 rather than Glu, binds zinc with a reduced affinity compared with native bovine alpha-LA, suggesting that the site identified from the X-ray structure did not correspond to strong zinc binding in solution.     

The structure of denatured alpha-lactalbumin elucidated by the technique of disulfide scrambling: fractionation of conformational isomers of alpha-lactalbumin

The structure of denatured alpha-lactalbumin (alpha-LA) has been characterized using the method of disulfide scrambling. Under denaturing conditions (urea, guanidine hydrochloride, guanidine thiocyanate, organic solvent or elevated temperature) and in the presence of thiol initiator, alpha-LA denatures by shuffling its four native disulfide bonds and converts to a mixture of fully oxidized scrambled structures. Analysis by reversed-phase HPLC reveals that the denatured alpha-LA comprises a minimum of 45 fractions of scrambled isomers. Among them, six well populated isomers have been isolated and structurally characterized. Their relative concentrations, which represent the fingerprinting of the denatured alpha-LA, vary substantially under different denaturing conditions. These results permit independent plotting of the denaturation and unfolding curves of alpha-LA. Most importantly, unique isomers of partially unfolded alpha-LA were shown to populate at mild and selected denaturing conditions. Organic solvent disrupts preferentially the hydrophobic alpha-helical domain, generating a predominant isomer containing two native disulfide bonds at the beta-sheet domain and two scrambled disulfide bonds at the alpha-helical region. Thermal denaturation selectively unfolds the beta-sheet domain of alpha-LA, producing a prevalent isomer that exhibits structural characteristics of the molten globule state of alpha-LA.     

Mutating aspartate in the calcium-binding site of alpha-lactalbumin: effects on the protein stability and cation binding.

The residue Asp87, which is in the calcium-binding loop of bovine alpha-lactalbumin (alpha-LA) and provides a side-chain carboxylate oxygen for ligand Ca(II) co-ordination, was substituted by either alanine or asparagine. The physical properties and calcium-binding affinities were monitored by intrinsic fluorescence and circular dichroism spectroscopy. D87A alpha-LA displayed a total loss of rigid tertiary structure, a dramatic loss in secondary structure and negligible calcium affinity [Anderson et al. (1997) Biochemistry, 36, 11648-11654]. On the contrary, D87N alpha-LA displayed native-like secondary structure with a somewhat de-stabilized tertiary structure. When the well-documented N-terminal methionine was enzymatically removed from D87N alpha-LA [Veprintsev et al. (1999) PROTEINS: Struct. Funct. Genet., 37, 65-72], the structure appeared to more closely resemble native alpha-LA. Remarkably, the thermal transition mid-temperature of apo-desMetD87N alpha-LA was approximately 31 degrees C versus native apo- alpha-LA (approximately 25 degrees C), probably due to negative charge 'compensation' in the calcium co-ordination site. On the other hand, the transition mid-temperature of Ca(II)-bound desMetD87N alpha-LA was approximately 57 degrees C versus native alpha-LA (approximately 66 degrees C), which was related to a decreased Ca(II) affinity (K = approximately 2.1 x 10(5) versus approximately 1.7 x 10(7)/M at 40 degrees C, respectively). These results reaffirm that alanine substitution in site specific mutagenesis is not always a prudent choice. Substitutions must be conservative with only minimal changes in functional groups and side-chain volume.     

Fusion of phospholipid vesicles induced by alpha-lactalbumin at acidic pH

Alpha-Lactalbumin (alpha-LA), lysozyme, and ribonuclease are found to induce fusion of phosphatidylserine/phosphatidylethanolamine vesicles at low pH. The fusogenic behavior and the binding to phospholipid vesicles of one of these proteins, alpha-LA, are studied at a wide range of conditions. The initial rate of fusion in the presence of alpha-LA increases with increasing acidity below pH 6, and the extent of alpha-LA binding to the vesicles is also found to increase with decreasing pH. Once bound to the vesicles in acidic media, the neutralization to pH 7 fails to dislodge the alpha-LA from the vesicles, and this irreversible binding also increases with decreasing pH. A segment of alpha-LA is found to be resistant to the proteolytic digestion when initially incubated with the vesicles at low pH. The amino acid composition of this fragment was determined, and from this the sequence of alpha-LA fragment, which appears to be inserted into the bilayer, is deduced. Hydrophobic labeling with dansyl chloride renders support that this segment indeed penetrates into the hydrophobic interior of bilayer. Since both the N-terminal and the C-terminal of this vesicle-bound protein are accessible to the externally added proteolytic enzymes, it is concluded that a loop of the polypeptide segment goes into the bilayer. These observations, taken together, suggest a possibility that the penetration by a loop of alpha-LA segment into the phospholipid bilayer is responsible for the fusion.     

Studies on cell-clearing activity in alpha-lactalbumin. Effects of calcium removal on activity and conformation.

Effects of calcium removal on the cell-clearing activity of alpha-lactalbumin (alpha-LA) and concomitant changes in conformational structure have been investigated as part of a continuing study of the activity found earlier [McKenzie, H.A. & White, F.H., Jr. (1987) Biochem. Int. 14, 347]. This activity is similar to that of lysozyme, whereby lysis of the bacterial cell wall is catalyzed. However, the specific activity of alpha-LA is on the order of 10(-6) that of lysozyme. Under conditions where activities of apo and native alpha-LA were approximately linear functions of the protein concentration, the maximal ratio of apo to native activity was 5.7:1, determined by comparison of second order velocity constants. The CD spectrum of apo alpha-LA is intermediate between that of the A state and the native protein. By NMR, the conformation of apo alpha-LA is similar to, but distinctly different from, that of the native protein. The apo form did not revert completely to the native state when Ca(II) was resupplied, consistent with a role for this cation in folding. It is suggested that the activity increase may result from a diminished constriction of the "cleft" region in alpha-LA.     

Limited proteolysis of bovine alpha-lactalbumin: isolation and characterization of protein domains

The partly folded states of alpha-lactalbumin (alpha-LA) exposed to acid solution at pH 2.0 (A-state) or at neutral pH upon EDTA-mediated removal of the single protein-bound calcium ion (apo form) have been probed by limited proteolysis experiments. These states are nowadays commonly considered to be molten globules and thus protein-folding intermediates. Pepsin was used for proteolysis at acid pH, while proteinase K and chymotrypsin at neutral pH. The expectations were that these proteolytic probes would detect sites and/or chain regions in the partly folded states of alpha-LA sufficiently dynamic, or even unfolded, capable of binding and adaptation to the specific stereochemistry of the protease's active site. A time-course analysis of the proteolytic events revealed that the fast, initial proteolytic cuts of the 123-residue chain of alpha-LA in its A-state or apo form by the three proteases occur at the same chain region 39-54, the actual site(s) of cleavage depending upon the protease employed. This region in native alpha-LA encompasses the beta-sheets of the protein. Subsequent cleavages occur mostly at chain regions 31-35 and 95-105. Four fragment species of alpha-LA have been isolated by reverse-phase high-performance liquid chromatography, and their conformational properties examined by circular dichroism and fluorescence emission spectroscopy. The single chain fragment 53-103, containing all the binding sites for calcium in native alpha-LA and cross-linked by two disulfide bridges, maintains in aqueous buffer and in the presence of calcium ions a folded structure characterized by the same content of alpha-helix of the corresponding chain segment in native alpha-LA. Evidence for some structure was also obtained for the two-chain species 1-40 and 104-123, as well as 1-31 and 105-123, both systems being covalently linked by two disulfide bonds. In contrast, the protein species given by fragment 1-34 connected to fragment 54-123 or 57-123 via four disulfide bridges adopts in solution a folded structure with the helical content expected for a native-like conformation. Of interest, the proteolytic fragment species herewith isolated correspond to the structural domains and subdomains of alpha-LA that can be identified by computational analysis of the three-dimensional structure of native alpha-LA (Siddiqui AS, Barton GI, 1995, Protein Sci 4:872-884). The fast, initial cleavages at the level of the beta-sheet region of native alpha-LA indicate that this region is highly mobile or even unfolded in the alpha-LA molten globule(s), while the rest of the protein chain maintains sufficient structure and rigidity to prevent extensive proteolysis. The subsequent cleavages at chain segment 95-105 indicate that also this region is somewhat mobile in the A-state or apo form of the protein. It is concluded that the overall domain topology of native alpha-LA is maintained in acid or at neutral pH upon calcium depletion. Moreover, the molecular properties of the partly folded states of alpha-LA deduced here from proteolysis experiments do correlate with those derived from previous NMR and other physicochemical measurements.     

Studies on a trace cell lytic activity associated with alpha-lactalbumin

alpha-Lactalbumin (alpha-LA) has been examined with a new and sensitive method for determination of lysozyme activity. Samples of bovine, human, equine, and rat alpha-LA exhibited cell lytic activity, from 2 X 10(-6) to 45 X 10(-6) of the specific activity of hen eggwhite lysozyme. The activity was chromatographically inseparable from bovine and human alpha-LA. Bovine serum albumin and purified beta-lactoglobulin were inactive. The pH profiles and reaction kinetics of bovine and human alpha-LA showed differences from those of the corresponding milk lysozymes, indicating that their lytic activities were not likely to have resulted from trace lysozyme content. Thus, it appears that a weak cell lytic activity is inherent to alpha-LA.     

Alpha-lipoic acid induces apoptosis in hepatoma cells via the PTEN/Akt pathway

We report here that alpha-lipoic acid (alpha-LA), a naturally-occurring antioxidant, scavenges reactive oxygen species (ROS) followed by an increase in apoptosis of human hepatoma cells. Apoptosis induced by alpha-LA was dependent upon the activation of the caspase cascade and the mitochondrial death pathway. alpha-LA induced increases in caspase-9 and caspase-3 but had no significant effect on caspase-8 activity. Apoptosis induced by alpha-LA was found to be mediated through the tensin homologue deleted on chromosome 10 (PTEN)/Akt pathway. Prior to cell apoptosis, PTEN was activated and its downstream target Akt was inhibited. Our findings indicate that increasing ROS scavenging could be a therapeutic strategy to treat cancer.     

Alpha-lactalbumin forms a compact molten globule in the absence of disulfide bonds.

Human alpha-lactalbumin (alpha-LA) is a four disulfide-bonded protein that adopts partially structured conformations under a variety of mildly denaturing conditions. At low pH, the protein is denatured but compact, with a high degree of secondary structure and a native-like fold. This is commonly referred to as a molten globule. A variant of alpha-LA, in which all eight cysteines have been mutated to alanine (all-Ala alpha-LA), has been studied using NMR spectroscopy. At low pH all-Ala alpha-LA is nearly as compact as wild type alpha-LA. Urea-induced unfolding experiments reveal that the residues that remain compact in the absence of disulfide bonds are those that are most resistant to unfolding in the wild-type alpha-LA molten globule. This is particularly remarkable because this stable core is formed by segments of the polypeptide chain from both the N- and C-termini. These results show that the overall architecture of the protein fold of alpha-LA is determined by the polypeptide sequence itself, and not as the result of cross-linking by disulfide bonds, and provide insight into the way in which the sequence codes for the fold.     

New biological function of bovine alpha-lactalbumin: protective effect against ethanol- and stress-induced gastric mucosal injury in rats.

Although several studies have shown that milk protein components have a wide range of biological activities, the potential role of these proteins in the gastrointestinal mucosal defense system is less well elucidated. In this study, we investigated the effect of the major proteins in cow's milk on gastric mucosal injury by using two acute ulcer models in Wistar rats. Gastric mucosal injury was induced by either intragastric 60% ethanol-HCl or water-immersion restraint stress (23 degrees C, 7 h). Each test milk protein was orally administered 30 min before the induction of gastric injury. Among the major milk proteins, alpha-lactalbumin (alpha-LA) is demonstrated to have a marked protective effect against ethanol-induced gastric injury, with the same potency as that of the typical antiulcer agent, Selbex. Whey protein isolate (WPI), which contained 25% alpha-LA, also protected against gastric injury, while casein showed no effect. Comparative studies on the protective effect of the four major components of WPI, beta-lactoglobulin, alpha-LA, bovine serum albumin and gamma-globulins (immunoglobulins), on the basis of their contents in WPI revealed that alpha-LA was responsible for the protective effect of WPI, being about 4-fold more effective than WPI itself. Alpha-LA showed dose-dependent protection against gastric injury induced by stress as well as ethanol. Pretreatment with indomethacin (10 mg/kg body weight, s.c.), which is a potent inhibitor of endogenous prostaglandin synthesis, resulted in a significant reduction in the protective effect of alpha-LA. These results indicate that alpha-LA has marked antiulcer activity as an active component of cow's milk protein, and suggest that alpha-LA intake may serve to protect against gastric mucosal injury, in part through endogenous prostaglandin synthesis.     

Stepwise proteolytic removal of the beta subdomain in alpha-lactalbumin. The protein remains folded and can form the molten globule in acid solution.

Bovine alpha-lactalbumin (alpha-LA) is an alpha/beta protein which adopts partly folded states when dissolved at low pH (A-state), by removal of the protein-bound calcium at neutral pH and low salt concentration (apo-state), as well as in aqueous trifluoroethanol. Previous spectroscopic studies have indicated that the A-state of alpha-LA at pH 2.0, considered a prototype molten globule, has a native-like fold in which the helical core is mostly retained, while the beta subdomain is less structured. Here, we investigate the conformational features of three derivatives of alpha-LA characterized by a single peptide bond fission or a deletion of 12 or 19/22 amino-acid residues of the beta subdomain of the native protein (approximately from residue 34 to 57). These alpha-LA derivatives were obtained by limited proteolysis of the protein in its partly folded state(s). A nicked alpha-LA species consisting of fragments 1-,3-40 and 41-123 (nicked-LA) was prepared by thermolytic digestion of the 123-residue chain of alpha-LA in 50% (v/v) aqueous trifluoroethanol. Two truncated or gapped protein species given by fragments 1-40 and 53-123 (desbeta1-LA) or fragments 1-34 and 54-,57-123 (desbeta2-LA) were obtained by digestion of alpha-LA with pepsin in acid or with proteinase K at neutral pH in its apo-state, respectively. The two protein fragments of nicked or gapped alpha-LA are covalently linked by the four disulfide bridges of the native protein. CD measurements revealed that, in aqueous solution at neutral pH and in the presence of calcium, the three protein species maintain the helical secondary structure of intact alpha-LA, while the tertiary structure is strongly affected by the proteolytic cleavages of the chain. Temperature effects of CD signals in the far- and near-UV region reveal a much more labile tertiary structure in the alpha-LA derivatives, while the secondary structure is mostly retained even upon heating. In acid solution at pH 2.0, the three alpha-LA variants adopt a conformational state essentially identical to the molten globule displayed by intact alpha-LA, as demonstrated by CD measurements. Moreover, they bind strongly the fluorescent dye 8-anilinonaphthalene-1-sulfonate, which is considered a diagnostic feature of the molten globule of proteins. Therefore, the beta subdomain can be removed from the alpha-LA molecule without impairing the capability of the rest of the chain to adopt a molten globule state. The results of this protein dissection study provide direct experimental evidence that in the alpha-LA molten globule only the alpha domain is structured.     

Electrostatic interactions in the acid denaturation of alpha-lactalbumin determined by NMR.

alpha-Lactalbumin (alpha-LA) undergoes a pH-dependent unfolding from the native state to a partially unfolded state (the molten globule state). To understand the role of electrostatic interactions in protein denaturation, NMR and CD pH titration experiments are performed on guinea pig alpha-LA. Variation of pH over the range of 7.0 to 2.0 simultaneously leads to the acid denaturation of the protein and the titration of individual ionizable groups. The pH titrations are interpreted in the context of these coupled events, and indicate that acid denaturation in alpha-LA is a cooperative event that is triggered by the protonation of two ionizable residues. Our NMR results suggest that the critical electrostatic interactions that contribute to the denaturation of alpha-LA are concentrated in the calcium binding region of the protein.     

Comparison of specific radioactivities of human alpha-lactalbumin iodinated by three different methods

Radioiodination provides an extremely sensitive method for the detection of low levels of proteins. In the development of a sensitive radioimmunoassay for human alpha-lactalbumin (alpha-LA), the protein was labeled to high specific activity (approaching 2000 Ci/mmol) with lactoperoxidase, chloramine-T, and Iodogen. Despite high specific activities of the labeled protein by each method, there was a considerable difference in their binding affinity with monoclonal anti-human alpha-LA antibodies due to varying degrees of protein damage. Iodination of human alpha-LA with Iodogen resulted in labels of the highest specific activity and immunoreactivity with the monoclonal antibodies used.     

Apo alpha-lactalbumin and lysozyme are colocalized in their subsequently formed spherical supramolecular assembly

We have reported previously that the calcium-depleted form of bovine alpha-lactalbumin (apo alpha-LA) interacts with hen egg-white lysozyme (LYS) to form spherical supramolecular structures. These supramolecular structures contain an equimolar ratio of the two proteins. We further explore here the organization of these structures. The spherical morphology and size of the assembled LYS/apo alpha-LA supramolecular structures were demonstrated using confocal scanning laser microscopy and scanning electron microscopy. From confocal scanning laser microscopy experiments with labelled proteins, it was found that LYS and apo alpha-LA were perfectly colocalized and homogeneously distributed throughout the entire three-dimensional structure of the microspheres formed. The spatial colocalization of the two proteins was also confirmed by the occurrence of a fluorescence resonance energy transfer phenomenon between labelled apo alpha-LA and labelled LYS. Polarized light microscopy analysis revealed that the microspheres formed differ from spherulites, a higher order semicrystalline structure. As the molecular mechanism initiating the formation of these microspheres is still unknown, we discuss the potential involvement of a LYS/apo alpha-LA heterodimer as a starting block for such a supramolecular assembly.