A critical evaluation of the predicted and X-ray structures of alpha-lactalbumin

The rapidly increasing availability of protein amino-acid sequences, many of which have been determined from the corresponding gene sequences, has intensified interest in the prediction of related protein structures when the three-dimensional structure of another member of the family is known. The study of bovine -Lactalbumin provides a classic example in which the three-dimensional structure was predicted, first by Browneet al. (1969) and later by Warmeet al. (1974), from the three-dimensional structure of hen-egg-white lysozyme (Blakeet al., 1965), taking into account the striking relationship between the amino acid sequences of the two proteins. A comprehensive comparison of these models with the structure of baboon -Lactalbumin derived from X-ray crystallography (Acharyaet al., 1989) is presented. The models mostly compare well with the experimentally determined structure except in the flexible C-terminal region of the molecule (rms deviation on C of residues 1–95, 1.1 Å).     

Identifying a Folding Nucleus for the Lysozyme/α-Lactalbumin Family from Sequence Conservation Clusters

Four subfamilies of c-type lysozyme and one subfamily of α-lactalbumin are defined from 78 sequences, and their folding nucleus is identified with a method based on conserved residues and native structural contacts between pairs of conserved residues. One large cluster of 19 conserved residues is found which is mostly nonpolar, buried, and nonfunctional. It can be subdivided into three subclusters: (1) conserved residues in four helices; (2) conserved residues that stabilize the connector between the α and the β domains; and (3) a β-turn, sitting in the middle of a bowl of α-helix residues. It is proposed that this folding nucleus initiates four helices, A, B, C, and D, three β sheets, and the connector, which corresponds closely to the nucleation of the so-called fast folding track pathway. As the secondary structures propagate, nonconserved residues and functionally conserved residues would form additional contacts. The conserved residues are selected with a phylogenetic scheme in which single members of subfamilies are selected. Subfamilies are then equally weighted to obtain the consensus conservation. Received: 11 June 2001 / Accepted: 28 August 2001     

Lipids as cofactors in protein folding: stereo-specific lipid-protein interactions are required to form HAMLET (human alpha-lactalbumin made lethal to tumor cells)

Proteins can adjust their structure and function in response to shifting environments. Functional diversity is created not only by the sequence but by changes in tertiary structure. Here we present evidence that lipid cofactors may enable otherwise unstable protein folding variants to maintain their conformation and to form novel, biologically active complexes. We have identified unsaturated C18 fatty acids in the cis conformation as the cofactors that bind apo alpha-lactalbumin and form HAMLET (human alpha-lactalbumin made lethal to tumor cells). The complexes were formed on an ion exchange column, were stable in a molten globule-like conformation, and had attained the novel biological activity. The protein-fatty acid interaction was specific, as saturated C18 fatty acids, or unsaturated C18:1trans conformers were unable to form complexes with apo alpha-lactalbumin, as were fatty acids with shorter or longer carbon chains. Unsaturated cis fatty acids other than C18:1:9cis were able to form stable complexes, but these were not active in the apoptosis assay. The results demonstrate that stereo-specific lipid-protein interactions can stabilize partially unfolded conformations and form molecular complexes with novel biological activity. The results offer a new mechanism for the functional diversity of proteins, by exploiting lipids as essential, tissue-specific cofactors in this process.     

A Novel Initiation Mechanism of Death in Streptococcus pneumoniae Induced by the Human Milk Protein-Lipid Complex HAMLET and Activated during Physiological Death

To cause colonization or infection, most bacteria grow in biofilms where differentiation and death of subpopulations is critical for optimal survival of the whole population. However, little is known about initiation of bacterial death under physiological conditions. Membrane depolarization has been suggested, but never shown to be involved, due to the difficulty of performing such studies in bacteria and the paucity of information that exists regarding ion transport mechanisms in prokaryotes. In this study, we performed the first extensive investigation of ion transport and membrane depolarization in a bacterial system. We found that HAMLET, a human milk protein-lipid complex, kills Streptococcus pneumoniae (the pneumococcus) in a manner that shares features with activation of physiological death from starvation. Addition of HAMLET to pneumococci dissipated membrane polarity, but depolarization per se was not enough to trigger death. Rather, both HAMLET- and starvation-induced death of pneumococci specifically required a sodium-dependent calcium influx, as shown using calcium and sodium transport inhibitors. This mechanism was verified under low sodium conditions, and in the presence of ionomycin or monensin, which enhanced pneumococcal sensitivity to HAMLET- and starvation-induced death. Pneumococcal death was also inhibited by kinase inhibitors, and indicated the involvement of Ser/Thr kinases in these processes. The importance of this activation mechanism was made evident, as dysregulation and manipulation of physiological death was detrimental to biofilm formation, a hallmark of bacterial colonization. Overall, our findings provide novel information on the role of ion transport during bacterial death, with the potential to uncover future antimicrobial targets.     

Proteomic analysis of hypoxia and non-hypoxia secretome mesenchymal stem-like cells from human breastmilk

IntroductionBreastmilk contains proteins and cells which have stem cell properties. The human breastmilk stem cell mimick mesenchymal stem cells and expresses pluripotency genes. The protein level of breastmilk is high in colostrum and gradually subsides in the first year of lactation. The mesenchymal stem cells from breastmilk can be an alternative source of stem cells that can potentially affect cardiovascular therapy. This study aimed to identify the proteomic analysis of secretome mesenchymal stem-like cells under hypoxia compared to non-hypoxia from human breastmilk stem cells.Material and methodsThe human breastmilk was collected from six healthy breastfeeding women and transported to the laboratory under aseptic conditions. The breastmilk cells were isolated then cultured. After 72 h, the human breastmilk stem cells reached confluence then cleaned up and isolated in serum-free media (spheroid) to allow serial passaging every 48 h. The acquisition stem cell was made with flow cytometry. The cells were divided into hBSC secretomes under hypoxia (A) and non-hypoxia (B) and analyzed for LC-MS to identify the peptide structure.ResultsThe human breastmilk cells contained several mesenchymal stem-like cells in density 2.4 × 10⁶ cell/mL for hypoxia and 2 × 10⁶ cell/mL for non-hypoxia conditions. The human breastmilk stem cell surface markers derived from the third cell passage process were 93.77% for CD44, 98.69% for CD73, 88.45% for CD90, and 96.30% for CD105. The protein level of secretome mesenchymal stem -like cells under hypoxia was measured at 5.56 μg/mL and 4.28 μg/mL for non-hypoxia. The liquid chromatography-mass spectrometry analysis identified 130 and 59 peptides from hypoxia and non-hypoxia of the human breastmilk stem cell secretome sequentially. Some important proteomics structures were found in the hypoxic human breastmilk stem cell secretome, such as transforming growth factor-β, VE-cadherin, and caspase.ConclusionThe human breastmilk cells contain mesenchymal stem-like cells and a high concentration of CD44, CD73, CD90, and CD105 as surface markers at third passage culture. The hypoxic hBSC secretome produces a higher protein level compare to non-hypoxia. The transforming growth factor -β was found in the hypoxic hBSC secretome as a modulator of VEGF-mediated angiogenesis.     

Conformational Isomers of Denatured and Unfolded Proteins: Methods of Production and Applications

Conformational isomers of denatured-unfolded proteins are rich in numbers and varied in shapes. They represent an opulent resource of biological molecules that have remained unexploited. The major obstacle in utilizing this untapped potential is that it is inherently difficult to isolate and characterize pure conformational isomers, not only because of the excessive large number, but also because of their instability and rapid inter-conversion. Our lab has developed a method for trapping selected conformational isomers of denatured proteins that are amenable to isolation, characterization and further applications. The method has potential usefulness, ranging from the comprehensive structural characterization of denatured proteins, to the elucidation of pathways of protein unfolding–folding, to the production of unlimited structurally defined non-native protein isomers for biomedical applications.     

Purification and characterization of mouse α-lactalbumin from lactating mammary glands

The whey protein, α-lactalbumin, was purified from lactating mammary glands of mice at high yields. It exists as two major charge forms (pI values of 6.2 and 5.8) with similar molecular weights (approx. 14 00). Antibodies prepared against these peptides precipitate newly synthesized and secreted α-lactalbumin from organ cultures of mid-pregnancy mammary glands. The antibody is specific for mouse α-lactalbumin as it does not react with mouse casein, mouse serum or purified bovine α-lactalbumin or galactosyl transferase. In addition, it blocks enzymatic activity of α-lactalbumin in mouse milk but has no effect on guinea pig or human milk. A very sensitive radioimmunoassay has been developed with this antibody which can detect α-lactalbumin levels as low as 0.25 ng.     

Hydrolysis of α-lactalbumin by cardosin A immobilized on highly activated supports

In the present research effort, production of derivatives of cardosin A (a plant protease) encompassing full stabilization of its dimeric structure has been achieved, via covalent, multi-subunit immobilization onto highly activated agarose-glutaraldehyde supports. Boiling such enzyme derivatives in the presence of sodium dodecyl sulfate and β-mercaptoethanol did not lead to leaching of enzyme, thus providing evidence for the effectiveness of the attachment procedure. Furthermore, the cardosin A derivatives prepared under optimal conditions presented ca. half the specific activity of the enzyme in soluble form, and were successfully employed at laboratory-scale trials to perform (selective) hydrolysis of α-lactalbumin (α-La), one of the major proteins in bovine whey. Hydrolysates of α-La were assayed for by the OPA method, as well as by FPLC, SDS–PAGE and HPLC. Thermal inactivation of the immobilized cardosin A was also assessed at 40, 50 and 55 °C; at these temperatures, no thermal denaturation took place during incubation for 48 h. The highest degree of hydrolysis was attained by 5 h reaction, at 55 °C and pH 5.2. SDS–PAGE of α-La hydrolysates displayed bands corresponding to low molecular weight peptides. Our results suggest that cardosin A in immobilized form is a good candidate to bring about proteolysis in the dairy industry, namely in whey processing.