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Catharina Svanborg - One of the best experts on this subject based on the ideXlab platform.
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α Lactalbumin engineered to be nonnative and inactive kills tumor cells when in complex with oleic acid a new biological function resulting from partial unfolding
Journal of Molecular Biology, 2009Co-Authors: Jenny Petterssonkastberg, Ann-kristin Mossberg, Catharina Svanborg, Maria Trulsson, Yeon Joong Yong, Soyoung Min, Yoongho Lim, John E ObrienAbstract:HAMLET (human alpha-Lactalbumin made lethal to tumor cells) is a tumoricidal complex consisting of partially unfolded protein and fatty acid and was first identified in casein fractions of human breast milk. The complex can be produced from its pure components through a modified chromatographic procedure where preapplied oleic acid binds with partially unfolded alpha-Lactalbumin on the stationary phase in situ. Because native alpha-Lactalbumin itself cannot trigger cell death, HAMLET's remarkable tumor-selective cytotoxicity has been strongly correlated with the conformational change of the protein upon forming the complex, but whether a recovery to the native state subsequently occurs upon entering the tumor cell is yet unclear. To this end, we utilize a recombinant variant of human alpha-Lactalbumin in which all eight cysteine residues are substituted for alanines (rHLA(all-Ala)), rendering the protein nonnative and biologically inactive under all conditions. The HAMLET analogue formed from the complex of rHLA(all-Ala) and oleic acid (rHLA(all-Ala)-OA) exhibited equivalent strong tumoricidal activity against lymphoma and carcinoma cell lines and was shown to accumulate within the nuclei of tumor cells, thus reproducing the cellular trafficking pattern of HAMLET. In contrast, the fatty acid-free rHLA(all-Ala) protein associated with the tumor cell surface but was not internalized and lacked any cytotoxic activity. Structurally, whereas HAMLET exhibited some residual native character in terms of NMR chemical shift dispersion, rHLA(all-Ala)-OA showed significant differences to HAMLET and, in fact, was found to be devoid of any tertiary packing. The results identify alpha-Lactalbumin as a protein with strikingly different functions in the native and partially unfolded states. We posit that partial unfolding offers another significant route of functional diversification for proteins within the cell.
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alpha Lactalbumin species variation hamlet formation and tumor cell death
Biochemical and Biophysical Research Communications, 2006Co-Authors: Jenny Pettersson, Ann-kristin Mossberg, Catharina SvanborgAbstract:HAMLET (human α-Lactalbumin made lethal to tumor cells) is a tumoricidal complex of apo α-Lactalbumin and oleic acid, formed in casein after low pH treatment of human milk. This study examined if HAMLET-like complexes are present in casein from different species and if isolated α-Lactalbumin from those species can form such complexes with oleic acid. Casein from human, bovine, equine, and porcine milk was separated by ion exchange chromatography and active complexes were only found in human casein. This was not explained by α-Lactalbumin sequence variation, as purified bovine, equine, porcine, and caprine α-Lactalbumins formed complexes with oleic acid with biological activity similar to HAMLET. We conclude that structural variation of α-Lactalbumins does not preclude the formation of HAMLET-like complexes and that natural HAMLET formation in casein was unique to human milk, which also showed the highest oleic acid content.
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stability of hamlet a kinetically trapped alpha Lactalbumin oleic acid complex
Protein Science, 2005Co-Authors: Jonas Fast, Ann-kristin Mossberg, Catharina Svanborg, Sara LinseAbstract:The stability toward thermal and urea denaturation was measured for HAMLET (human α-Lactalbumin made lethal to tumor cells) and α-Lactalbumin, using circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Under all conditions examined, HAMLET appears to have the same or lower stability than α-Lactalbumin. The largest difference is seen for thermal denaturation of the calcium free (apo) forms, where the temperature at the transition midpoint is 15°C lower for apo HAMLET than for apo α-Lactalbumin. The difference becomes progressively smaller as the calcium concentration increases. Denaturation of HAMLET was found to be irreversible. Samples of HAMLET that have been renatured after denaturation have lost the specific biological activity toward tumor cells. Three lines of evidence indicate that HAMLET is a kinetic trap: (1) It has lower stability than α-Lactalbumin, although it is a complex of α-Lactalbumin and oleic acid; (2) its denaturation is irreversible and HAMLET is lost after denaturation; (3) formation of HAMLET requires a specific conversion protocol.
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treatment of skin papillomas with topical alpha Lactalbumin oleic acid
The New England Journal of Medicine, 2004Co-Authors: Lotta Gustafsson, Ann-kristin Mossberg, Irene Leijonhufvud, Annika Aronsson, Catharina SvanborgAbstract:background We studied the effect on skin papillomas of topical application of a complex of a -Lactalbumin and oleic acid (often referred to as human a -Lactalbumin made lethal to tumor cells [HAMLET]) to establish proof of the principle that a -Lactalbumin–oleic acid kills transformed cells but not healthy, differentiated cells. methods Forty patients with cutaneous papillomas that were resistant to conventional treatment were enrolled in a randomized, placebo-controlled, double-blind study, in which a -Lactalbumin–oleic acid or saline placebo was applied daily for three weeks and the change in the volume of each lesion was recorded. After this first phase of the study, 34 patients participated in the second phase, an open-label trial of a three-week course of a -Lactalbumin–oleic acid. Approximately two years after the end of the open-label phase of the study, 38 of the original 40 patients were examined, and long-term followup data were obtained. results In the first phase of the study, the lesion volume was reduced by 75 percent or more in all 20 patients in the a -Lactalbumin–oleic acid group, and in 88 of 92 papillomas; in the placebo group, a similar effect was evident in only 3 of 20 patients (15 of 74 papillomas) (P<0.001). After the patients in the initial placebo group had been treated with a -Lactalbumin–oleic acid in the second phase of the study, a median reduction of 82 percent in lesion volume was observed. At follow-up two years after the end of the second phase, all lesions had completely resolved in 83 percent of the patients treated with a -Lactalbumin–oleic acid, and the time to resolution was shorter in the group originally assigned to receive a -Lactalbumin–oleic acid than among patients originally in the placebo group (2.4 vs. 9.9 months; P<0.01). No adverse reactions were reported, and there was no difference in the outcomes of treatment between immunocompetent and immunosuppressed patients. conclusions Treatment with topical a -Lactalbumin–oleic acid has a beneficial and lasting effect on skin papillomas.
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human alpha Lactalbumin made lethal to tumor cells hamlet kills human glioblastoma cells in brain xenografts by an apoptosis like mechanism and prolongs survival
Cancer Research, 2004Co-Authors: Walter Fischer, Ann-kristin Mossberg, Lotta Gustafsson, Janne Gronli, Sverre Mork, Rolf Bjerkvig, Catharina SvanborgAbstract:Malignant brain tumors present a major therapeutic challenge because no selective or efficient treatment is available. Here, we demonstrate that intratumoral administration of human α-Lactalbumin made lethal to tumor cells (HAMLET) prolongs survival in a human glioblastoma (GBM) xenograft model, by selective induction of tumor cell apoptosis. HAMLET is a protein-lipid complex that is formed from α-Lactalbumin when the protein changes its tertiary conformation and binds oleic acid as a cofactor. HAMLET induces apoptosis in a wide range of tumor cells in vitro, but the therapeutic effect in vivo has not been examined. In this study, invasively growing human GBM tumors were established in nude rats (Han:rnu/rnu Rowett, n = 20) by transplantation of human GBM biopsy spheroids. After 7 days, HAMLET was administered by intracerebral convection-enhanced delivery for 24 h into the tumor area; and α-Lactalbumin, the native, folded variant of the same protein, was used as a control. HAMLET reduced the intracranial tumor volume and delayed the onset of pressure symptoms in the tumor-bearing rats. After 8 weeks, all α-Lactalbumin-treated rats had developed pressure symptoms, but the HAMLET-treated rats remained asymptomatic. Magnetic resonance imaging scans revealed large differences in tumor volume (456 versus 63 mm3). HAMLET caused apoptosis in vivo in the tumor but not in adjacent intact brain tissue or in nontransformed human astrocytes, and no toxic side effects were observed. The results identify HAMLET as a new candidate in cancer therapy and suggest that HAMLET should be additionally explored as a novel approach to controlling GBM progression.
Ann-kristin Mossberg - One of the best experts on this subject based on the ideXlab platform.
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α Lactalbumin engineered to be nonnative and inactive kills tumor cells when in complex with oleic acid a new biological function resulting from partial unfolding
Journal of Molecular Biology, 2009Co-Authors: Jenny Petterssonkastberg, Ann-kristin Mossberg, Catharina Svanborg, Maria Trulsson, Yeon Joong Yong, Soyoung Min, Yoongho Lim, John E ObrienAbstract:HAMLET (human alpha-Lactalbumin made lethal to tumor cells) is a tumoricidal complex consisting of partially unfolded protein and fatty acid and was first identified in casein fractions of human breast milk. The complex can be produced from its pure components through a modified chromatographic procedure where preapplied oleic acid binds with partially unfolded alpha-Lactalbumin on the stationary phase in situ. Because native alpha-Lactalbumin itself cannot trigger cell death, HAMLET's remarkable tumor-selective cytotoxicity has been strongly correlated with the conformational change of the protein upon forming the complex, but whether a recovery to the native state subsequently occurs upon entering the tumor cell is yet unclear. To this end, we utilize a recombinant variant of human alpha-Lactalbumin in which all eight cysteine residues are substituted for alanines (rHLA(all-Ala)), rendering the protein nonnative and biologically inactive under all conditions. The HAMLET analogue formed from the complex of rHLA(all-Ala) and oleic acid (rHLA(all-Ala)-OA) exhibited equivalent strong tumoricidal activity against lymphoma and carcinoma cell lines and was shown to accumulate within the nuclei of tumor cells, thus reproducing the cellular trafficking pattern of HAMLET. In contrast, the fatty acid-free rHLA(all-Ala) protein associated with the tumor cell surface but was not internalized and lacked any cytotoxic activity. Structurally, whereas HAMLET exhibited some residual native character in terms of NMR chemical shift dispersion, rHLA(all-Ala)-OA showed significant differences to HAMLET and, in fact, was found to be devoid of any tertiary packing. The results identify alpha-Lactalbumin as a protein with strikingly different functions in the native and partially unfolded states. We posit that partial unfolding offers another significant route of functional diversification for proteins within the cell.
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alpha Lactalbumin species variation hamlet formation and tumor cell death
Biochemical and Biophysical Research Communications, 2006Co-Authors: Jenny Pettersson, Ann-kristin Mossberg, Catharina SvanborgAbstract:HAMLET (human α-Lactalbumin made lethal to tumor cells) is a tumoricidal complex of apo α-Lactalbumin and oleic acid, formed in casein after low pH treatment of human milk. This study examined if HAMLET-like complexes are present in casein from different species and if isolated α-Lactalbumin from those species can form such complexes with oleic acid. Casein from human, bovine, equine, and porcine milk was separated by ion exchange chromatography and active complexes were only found in human casein. This was not explained by α-Lactalbumin sequence variation, as purified bovine, equine, porcine, and caprine α-Lactalbumins formed complexes with oleic acid with biological activity similar to HAMLET. We conclude that structural variation of α-Lactalbumins does not preclude the formation of HAMLET-like complexes and that natural HAMLET formation in casein was unique to human milk, which also showed the highest oleic acid content.
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stability of hamlet a kinetically trapped alpha Lactalbumin oleic acid complex
Protein Science, 2005Co-Authors: Jonas Fast, Ann-kristin Mossberg, Catharina Svanborg, Sara LinseAbstract:The stability toward thermal and urea denaturation was measured for HAMLET (human α-Lactalbumin made lethal to tumor cells) and α-Lactalbumin, using circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Under all conditions examined, HAMLET appears to have the same or lower stability than α-Lactalbumin. The largest difference is seen for thermal denaturation of the calcium free (apo) forms, where the temperature at the transition midpoint is 15°C lower for apo HAMLET than for apo α-Lactalbumin. The difference becomes progressively smaller as the calcium concentration increases. Denaturation of HAMLET was found to be irreversible. Samples of HAMLET that have been renatured after denaturation have lost the specific biological activity toward tumor cells. Three lines of evidence indicate that HAMLET is a kinetic trap: (1) It has lower stability than α-Lactalbumin, although it is a complex of α-Lactalbumin and oleic acid; (2) its denaturation is irreversible and HAMLET is lost after denaturation; (3) formation of HAMLET requires a specific conversion protocol.
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treatment of skin papillomas with topical alpha Lactalbumin oleic acid
The New England Journal of Medicine, 2004Co-Authors: Lotta Gustafsson, Ann-kristin Mossberg, Irene Leijonhufvud, Annika Aronsson, Catharina SvanborgAbstract:background We studied the effect on skin papillomas of topical application of a complex of a -Lactalbumin and oleic acid (often referred to as human a -Lactalbumin made lethal to tumor cells [HAMLET]) to establish proof of the principle that a -Lactalbumin–oleic acid kills transformed cells but not healthy, differentiated cells. methods Forty patients with cutaneous papillomas that were resistant to conventional treatment were enrolled in a randomized, placebo-controlled, double-blind study, in which a -Lactalbumin–oleic acid or saline placebo was applied daily for three weeks and the change in the volume of each lesion was recorded. After this first phase of the study, 34 patients participated in the second phase, an open-label trial of a three-week course of a -Lactalbumin–oleic acid. Approximately two years after the end of the open-label phase of the study, 38 of the original 40 patients were examined, and long-term followup data were obtained. results In the first phase of the study, the lesion volume was reduced by 75 percent or more in all 20 patients in the a -Lactalbumin–oleic acid group, and in 88 of 92 papillomas; in the placebo group, a similar effect was evident in only 3 of 20 patients (15 of 74 papillomas) (P<0.001). After the patients in the initial placebo group had been treated with a -Lactalbumin–oleic acid in the second phase of the study, a median reduction of 82 percent in lesion volume was observed. At follow-up two years after the end of the second phase, all lesions had completely resolved in 83 percent of the patients treated with a -Lactalbumin–oleic acid, and the time to resolution was shorter in the group originally assigned to receive a -Lactalbumin–oleic acid than among patients originally in the placebo group (2.4 vs. 9.9 months; P<0.01). No adverse reactions were reported, and there was no difference in the outcomes of treatment between immunocompetent and immunosuppressed patients. conclusions Treatment with topical a -Lactalbumin–oleic acid has a beneficial and lasting effect on skin papillomas.
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human alpha Lactalbumin made lethal to tumor cells hamlet kills human glioblastoma cells in brain xenografts by an apoptosis like mechanism and prolongs survival
Cancer Research, 2004Co-Authors: Walter Fischer, Ann-kristin Mossberg, Lotta Gustafsson, Janne Gronli, Sverre Mork, Rolf Bjerkvig, Catharina SvanborgAbstract:Malignant brain tumors present a major therapeutic challenge because no selective or efficient treatment is available. Here, we demonstrate that intratumoral administration of human α-Lactalbumin made lethal to tumor cells (HAMLET) prolongs survival in a human glioblastoma (GBM) xenograft model, by selective induction of tumor cell apoptosis. HAMLET is a protein-lipid complex that is formed from α-Lactalbumin when the protein changes its tertiary conformation and binds oleic acid as a cofactor. HAMLET induces apoptosis in a wide range of tumor cells in vitro, but the therapeutic effect in vivo has not been examined. In this study, invasively growing human GBM tumors were established in nude rats (Han:rnu/rnu Rowett, n = 20) by transplantation of human GBM biopsy spheroids. After 7 days, HAMLET was administered by intracerebral convection-enhanced delivery for 24 h into the tumor area; and α-Lactalbumin, the native, folded variant of the same protein, was used as a control. HAMLET reduced the intracranial tumor volume and delayed the onset of pressure symptoms in the tumor-bearing rats. After 8 weeks, all α-Lactalbumin-treated rats had developed pressure symptoms, but the HAMLET-treated rats remained asymptomatic. Magnetic resonance imaging scans revealed large differences in tumor volume (456 versus 63 mm3). HAMLET caused apoptosis in vivo in the tumor but not in adjacent intact brain tissue or in nontransformed human astrocytes, and no toxic side effects were observed. The results identify HAMLET as a new candidate in cancer therapy and suggest that HAMLET should be additionally explored as a novel approach to controlling GBM progression.
Jingshun Zhang - One of the best experts on this subject based on the ideXlab platform.
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multiple reaction monitoring based determination of bovine α Lactalbumin in infant formulas and whey protein concentrates by ultra high performance liquid chromatography tandem mass spectrometry using tryptic signature peptides and synthetic peptide
Analytica Chimica Acta, 2012Co-Authors: Jingshun Zhang, Baifen Huang, Shiyun Lai, Yu Zhang, Yiping RenAbstract:The determination of α-Lactalbumin in various dairy products attracts wide attention in multidiscipline fields because of its nutritional and biological functions. In the present study, we quantified the bovine α-Lactalbumin in various infant formulas and whey protein concentrates using ultra-high performance liquid chromatography coupled to tandem mass spectrometer in multiple reaction monitoring mode. Bovine α-Lactalbumin was quantified by employing the synthetic internal standard based on the molar equivalent relationship among the internal standard, bovine α-Lactalbumin and their signature peptides. This study especially focused on the recovery rates of the sample preparation procedure and robust quantification of total bovine α-Lactalbumin in its native and thermally denatured form with a synthetic internal standard KILDKVGINNYWLAHKALCSE. The observed recovery rates of bovine α-Lactalbumin ranged from 95.8 to 100.6% and the reproducibility was excellent (RSD<6%) at different spiking levels. The limit of quantitation is 10 mg/100 g for infant formulas and whey protein concentrates. In order to validate the applicability of the method, 21 brands of infant formulas were analyzed. The acquired contents of bovine α-Lactalbumin were 0.67-1.84 g/100g in these infant formulas in agreement with their label claimed values. The experiment of heat treatment time showed that the loss of native α-Lactalbumin enhanced with an increasing intensity of heat treatment. Comparing with Ren's previous method by analysis of only native bovine α-Lactalbumin, the present method at the peptide level proved to be highly suitable for measuring bovine α-Lactalbumin in infant formulas and whey protein concentrates, avoiding forgoing the thermally induced denatured α-Lactalbumin caused by the technological processing.
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multiple reaction monitoring based determination of bovine α Lactalbumin in infant formulas and whey protein concentrates by ultra high performance liquid chromatography tandem mass spectrometry using tryptic signature peptides and synthetic peptide standards
Analytica Chimica Acta, 2012Co-Authors: Jingshun Zhang, Baifen Huang, Yu Zhang, Duo LiAbstract:Abstract The determination of α-Lactalbumin in various dairy products attracts wide attention in multidiscipline fields because of its nutritional and biological functions. In the present study, we quantified the bovine α-Lactalbumin in various infant formulas and whey protein concentrates using ultra-high performance liquid chromatography coupled to tandem mass spectrometer in multiple reaction monitoring mode. Bovine α-Lactalbumin was quantified by employing the synthetic internal standard based on the molar equivalent relationship among the internal standard, bovine α-Lactalbumin and their signature peptides. This study especially focused on the recovery rates of the sample preparation procedure and robust quantification of total bovine α-Lactalbumin in its native and thermally denatured form with a synthetic internal standard KILDKVGINNYWLAHKALCSE. The observed recovery rates of bovine α-Lactalbumin ranged from 95.8 to 100.6% and the reproducibility was excellent (RSD
Sara Linse - One of the best experts on this subject based on the ideXlab platform.
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stability of hamlet a kinetically trapped alpha Lactalbumin oleic acid complex
Protein Science, 2005Co-Authors: Jonas Fast, Ann-kristin Mossberg, Catharina Svanborg, Sara LinseAbstract:The stability toward thermal and urea denaturation was measured for HAMLET (human α-Lactalbumin made lethal to tumor cells) and α-Lactalbumin, using circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Under all conditions examined, HAMLET appears to have the same or lower stability than α-Lactalbumin. The largest difference is seen for thermal denaturation of the calcium free (apo) forms, where the temperature at the transition midpoint is 15°C lower for apo HAMLET than for apo α-Lactalbumin. The difference becomes progressively smaller as the calcium concentration increases. Denaturation of HAMLET was found to be irreversible. Samples of HAMLET that have been renatured after denaturation have lost the specific biological activity toward tumor cells. Three lines of evidence indicate that HAMLET is a kinetic trap: (1) It has lower stability than α-Lactalbumin, although it is a complex of α-Lactalbumin and oleic acid; (2) its denaturation is irreversible and HAMLET is lost after denaturation; (3) formation of HAMLET requires a specific conversion protocol.
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alpha Lactalbumin unfolding is not sufficient to cause apoptosis but is required for the conversion to hamlet human alpha Lactalbumin made lethal to tumor cells
Protein Science, 2003Co-Authors: Malin Svensson, Ann-kristin Mossberg, Sara Linse, Lotta Gustafsson, Lawrence J Berliner, Jonas Fast, Caroline Duringer, Oskar Hallgren, Charles L Brooks, Catharina SvanborgAbstract:HAMLET (human α-Lactalbumin made lethal to tumor cells) is a complex of human α-Lactalbumin and oleic acid (C18:1:9 cis) that kills tumor cells by an apoptosis-like mechanism. Previous studies have shown that a conformational change is required to form HAMLET from α-Lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of α-Lactalbumin is sufficient to induce cell death. We used the bovine α-Lactalbumin Ca2+ site mutant D87A, which is unable to bind Ca2+, and thus remains partially unfolded regardless of solvent conditions. The D87A mutant protein was found to be inactive in the apoptosis assay, but could readily be converted to a HAMLET-like complex in the presence of oleic acid. BAMLET (bovine α-Lactalbumin made lethal to tumor cells) and D87A-BAMLET complexes were both able to kill tumor cells. This activity was independent of the Ca2+site, as HAMLET maintained a high affinity for Ca2+ but D87A-BAMLET was active with no Ca2+ bound. We conclude that partial unfolding of α-Lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca2+-binding site is not required for conversion of α-Lactalbumin to the active complex or to cause cell death. This suggests that the lipid cofactor stabilizes the altered fold without interfering with the Ca2+site.
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Conversion of alpha-Lactalbumin to a protein inducing apoptosis.
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Malin Svensson, Ann-kristin Mossberg, Sara Linse, Anders Håkansson, Catharina SvanborgAbstract:In this study α-Lactalbumin was converted from the regular, native state to a folding variant with altered biological function. The folding variant was shown to induce apoptosis in tumor cells and immature cells, but healthy cells were resistant to this effect. Conversion to HAMLET (human α-Lactalbumin made lethal to tumor cells) required partial unfolding of the protein and a specific fatty acid, C18:1, as a necessary cofactor. Conversion was achieved with α-Lactalbumin derived from human milk whey and with recombinant protein expressed in Escherichia coli. We thus have identified the folding change and the fatty acid as two key elements that define HAMLET, the apoptosis-inducing functional state of α-Lactalbumin. Although the environment in the mammary gland favors the native conformation of α-Lactalbumin that serves as a specifier in the lactose synthase complex, the conditions under which HAMLET was formed resemble those in the stomach of the nursing child. Low pH is known to release Ca2+ from the high-affinity Ca2+-binding site and to activate lipases that hydrolyze free fatty acids from milk triglycerides. We propose that this single amino acid polypeptide chain may perform vastly different biological functions depending on its folding state and the in vivo environment. It may be speculated that molecules like HAMLET can aid in lowering the incidence of cancer in breast-fed children by purging of tumor cells from the gut of the neonate.
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a folding variant of alpha Lactalbumin with bactericidal activity against streptococcus pneumoniae
Molecular Microbiology, 2000Co-Authors: Anders Håkansson, Malin Svensson, Ann-kristin Mossberg, Sara Linse, Bo Lönnerdal, Hemant Sabharwal, Irene Lazou, Catharina SvanborgAbstract:This study describes an alpha-Lactalbumin folding variant from human milk with bactericidal activity against antibiotic-resistant and -susceptible strains of Streptococcus pneumoniae. The active complex precipitated with the casein fraction at pH 4.6 and was purified from casein by a combination of anion exchange and gel chromatography. Unlike other casein components, the active complex was retained on the ion-exchange matrix and eluted only with high salt. The eluted fraction showed N-terminal and mass spectrometric identity with human milk alpha-Lactalbumin, but native alpha-Lactalbumin had no bactericidal effect. Spectroscopic analysis demonstrated that the active form of the molecule was in a different folding state, with secondary structure identical to alpha-Lactalbumin from human milk whey, but fluctuating tertiary structure. Native alpha-Lactalbumin could be converted to the active bactericidal form by ion-exchange chromatography in the presence of a cofactor from human milk casein, characterized as a C18:1 fatty acid. Analysis of the antibacterial spectrum showed selectivity for streptococci; Gram-negative and other Gram-positive bacteria were resistant. The folding variant of alpha-Lactalbumin is a new example of naturally occurring molecules with antimicrobial activity.
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molecular characterization of alpha Lactalbumin folding variants that induce apoptosis in tumor cells
Journal of Biological Chemistry, 1999Co-Authors: Malin Svensson, Ann-kristin Mossberg, Catharina Svanborg, Anders Håkansson, Hemant Sabharwal, Peter H Lipniunas, Hakon Leffler, Sara LinseAbstract:This study characterized a protein complex in human milk that induces apoptosis in tumor cells but spares healthy cells. The active fraction was purified from casein by anion exchange chromatography. Unlike other casein components the active fraction was retained by the ion exchanger and eluted after a high salt gradient. The active fraction showed N-terminal amino acid sequence identity with human milk alpha-Lactalbumin and mass spectrometry ruled out post-translational modifications. Size exclusion chromatography resolved monomers and oligomers of alpha-Lactalbumin that were characterized using UV absorbance, fluorescence, and circular dichroism spectroscopy. The high molecular weight oligomers were kinetically stable against dissociation into monomers and were found to have an essentially retained secondary structure but a less well organized tertiary structure. Comparison with native monomeric and molten globule alpha-Lactalbumin showed that the active fraction contains oligomers of alpha-Lactalbumin that have undergone a conformational switch toward a molten globule-like state. Oligomerization appears to conserve alpha-Lactalbumin in a state with molten globule-like properties at physiological conditions. The results suggest differences in biological properties between folding variants of alpha-Lactalbumin.
Yiping Ren - One of the best experts on this subject based on the ideXlab platform.
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multiple reaction monitoring based determination of bovine α Lactalbumin in infant formulas and whey protein concentrates by ultra high performance liquid chromatography tandem mass spectrometry using tryptic signature peptides and synthetic peptide
Analytica Chimica Acta, 2012Co-Authors: Jingshun Zhang, Baifen Huang, Shiyun Lai, Yu Zhang, Yiping RenAbstract:The determination of α-Lactalbumin in various dairy products attracts wide attention in multidiscipline fields because of its nutritional and biological functions. In the present study, we quantified the bovine α-Lactalbumin in various infant formulas and whey protein concentrates using ultra-high performance liquid chromatography coupled to tandem mass spectrometer in multiple reaction monitoring mode. Bovine α-Lactalbumin was quantified by employing the synthetic internal standard based on the molar equivalent relationship among the internal standard, bovine α-Lactalbumin and their signature peptides. This study especially focused on the recovery rates of the sample preparation procedure and robust quantification of total bovine α-Lactalbumin in its native and thermally denatured form with a synthetic internal standard KILDKVGINNYWLAHKALCSE. The observed recovery rates of bovine α-Lactalbumin ranged from 95.8 to 100.6% and the reproducibility was excellent (RSD<6%) at different spiking levels. The limit of quantitation is 10 mg/100 g for infant formulas and whey protein concentrates. In order to validate the applicability of the method, 21 brands of infant formulas were analyzed. The acquired contents of bovine α-Lactalbumin were 0.67-1.84 g/100g in these infant formulas in agreement with their label claimed values. The experiment of heat treatment time showed that the loss of native α-Lactalbumin enhanced with an increasing intensity of heat treatment. Comparing with Ren's previous method by analysis of only native bovine α-Lactalbumin, the present method at the peptide level proved to be highly suitable for measuring bovine α-Lactalbumin in infant formulas and whey protein concentrates, avoiding forgoing the thermally induced denatured α-Lactalbumin caused by the technological processing.
