Guinea Pig Alpha-lactalbumin Research Articles

Production of correctly folded recombinant [13C, 15N]-enriched guinea pig [Val90]-alpha-lactalbumin.

The M90V mutant of guinea pig alpha-lactalbumin (gpLA) was expressed intracellularly in Escherichia coli using a gpLA gene fusion to the IgG-binding ('Z') domain coding sequences of staphylococcal protein A. The fusion protein was expressed as an inclusion body, then purified and refolded in vitro; CNBr cleavage of the fusion polypeptide yielded native alpha-lactalbumin. The recombinant M90V gpLA was virtually identical with natural gpLA with respect to its ability to stimulate lactose synthesis by galactosyl transferase and the recombinant and natural molecules also exhibited similar circular dichroism spectra, thermal melting profiles and NMR spectra. However, modest perturbations in the chemical shifts of amide protons in the C-helix residues, attributable to the Met-->Val mutation, were observed. In defined media, this expression system enabled the production of highly-enriched 15N- and 13C, 15N-labeled gpLA. Use of this material will allow the solution conformations of the native and molten globule states of gpLA to be characterized by high-resolution multidimensional NMR.

Temperature dependence of the phosphorescence quantum yield of various alpha-lactalbumins and of hen egg-white lysozyme

The radiative quantum yield, phi op, of the triplet state of human alpha-lactalbumin (HLA) has been measured in the temperature range between 6 K and the softening point of the aqueous glass (approximately 150 K). phi op has little temperature dependence below approximately 30 K, but above this it decreases sharply with increasing temperature. The unusual temperature dependence is fitted by a phenomenological two-state model in which the phosphorescence originates primarily from a donor, tryptophan (Trp) 104, and an acceptor, Trp 60, the populations of which are coupled by a thermally activated triplet-triplet energy transfer process. The model assumes that the acceptor (Trp 60) triplet state undergoes radiationless deactivation by a proximal disulfide residue, while the donor (Trp 104) has no such extrinsic quencher. The decrease of phi op with increasing temperature is accounted for by the thermally activated triplet-triplet energy transfer process. The disulfide quenching rate constant itself is assumed to be temperature independent, in accord with recent measurements of simple disulfide quenching in long chain snake venom neurotoxins (Schlyer, B. D., E. Lau, and A. H. Maki. 1992. Biochemistry. 31:4375-4383; Li, Z., A. Bruce, and W. C. Galley. 1992. Biophys. J. 61:1364-1371). We find that the phosphorescence quenching in HLA occurs with an activation energy of 97 cm-1, which we associate with a barrier to the energy transfer process. The data are fit well by the model if we assume a value for the temperature-independent disulfide quenching constant of kQ > 3 s-1 that is consistent with recent measurements on indole-disulfide model systems (Li, Z., A. Bruce, and W. C. Galley. 1992. Biophys. J. 61:1364-1371). Similar results are reported for bovine alpha-lactalbumin (BLA) and for hen egg-white lysozyme (HEWL) that contains the structural equivalents of Trp 104 and Trp 60 of HLA. HLA provides the best agreement with calculations since it is the simplest, lacking Trp 26, a residue not considered in the model, that probably contributes significantly to the phosphorescence of BLA, guinea pig alpha-lactalbumin (GPLA), and HEWL. GPLA, which contains Trp 104 but lacks Trp 60, shows qualitatively less thermally induced phosphorescence quenching than HLA, BLA, and HEWL, thus supporting the postulated quenching model.

Structure and stability of the molten globule state of guinea-pig alpha-lactalbumin: a hydrogen exchange study.

A partially folded state of guinea pig alpha-lactalbumin (the A-state or molten globule state), formed by denaturation at low pH, has been studied using hydrogen exchange methods. The overall distribution of exchange kinetics, measured by 1-D NMR, suggests that fewer than 20 amides in the structure are involved in highly persistent residual structure, although CD results suggest that many other parts of the chain are folded, for a significant proportion of the time, into less stable structural elements. The pH-jump experiments show that some amides that are strongly protected from exchange in the native state become freely accessible in the A-state but that conversely a majority, at least, of those that are slow to exchange in the A-state retain that protection in the native state. This suggests that the persistent structure in the A-state is native-like although the possibility that nonnative like structural elements persist cannot be eliminated. Resonance assignments for key residues in the NMR spectrum of the native state have enabled us to use the pH-jump method also to identify the majority of the most protected amides in the A-state: they are located in two hydrophobic segments, corresponding to the B- and C-helices of the native structure. This strongly suggests that the most persistent structure of the A-state includes these regions. A variety of lines of evidence, including fluorescence quenching data and, most remarkably, very effective protection from exchange of an indole NH in a tryptophan side chain, suggest that some form of hydrophobic core in the helical domain of the native structure persists in the A-state, although without the stereochemical rigidity of the native tertiary structure. The other domain of the native structure, including the beta-sheet, appears not to contain structural elements which persist to the same extent in the A-state, emphasizing that the molten globule is highly heterogeneous, in terms of the stability and specificity of both backbone and side chain interactions.

Transgenic mice carrying the guinea-pig α-lactalbumin gene transcribe milk protein genes in their sebaceous glands during lactation

We have generated transgenic mice carrying the entire guinea-pig alpha-lactalbumin gene. Lactating transgenic mice expressed high levels of correctly initiated and processed guinea-pig alpha-lactalbumin mRNA in the secretory epithelium of their mammary glands, and secreted guinea-pig alpha-lactalbumin in their milk. Transcripts were detectable after 7 days of pregnancy, indicating that the transgene was under correct hormonal control. Whereas no or negligible transcription was detectable in all other tissues tested, high levels of transcripts were found in the skin of lactating transgenic mice. Guinea-pig alpha-lactalbumin protein was undetectable in the skin, however. In situ hybridization analysis showed that expression was localized to the undifferentiated cells in the basal layer of the sebaceous glands. Further studies revealed high levels of endogenous beta-casein mRNA in normal lactating mouse skin, demonstrating that the transcription of milk protein genes in lactating mouse skin is a normal event, and is not peculiar to the transgene. This surprising finding highlights the developmental relationship of the mammary gland to other specialized structures of the skin, supports a role for epithelial-extracellular matrix interactions in the regulation of milk protein gene expression in vivo, and identifies the skin as a particularly accessible model system in which to study the regulation of milk protein gene expression. In addition, the guinea-pig alpha-lactalbumin gene will be a source of regulatory sequences with which to direct heterologous gene expression to the sebaceous glands of transgenic mice.

Hydrogen exchange of the tryptophan residues in bovine, goat, guinea pig, and human .alpha.-lactalbumin

Hydrogen exchange of the individual tryptophan residues of bovine, goat, guinea pig, and human alpha-lactalbumin has been studied by both ultraviolet and NMR spectra. The assignment of the slowly exchanging imino proton resonances to the tryptophan residues (Trp26 and Trp60) was obtained by comparison of the nuclear Overhauser effect difference spectra of bovine, guinea pig, and human alpha-lactalbumin. Taking account of the thermal unfolding of each alpha-lactalbumin, the hydrogen exchange rates of the individual tryptophan residues are analyzed. The temperature dependence of the exchange rates classified their exchange mechanisms into two exchange processes: the "low activation energy process" and the "high activation energy process" which is associated directly with the global thermal unfolding of the protein. Trp26 of alpha-lactalbumin exchanges through the high activation energy process. The exchange behavior of Trp26 of guinea pig alpha-lactalbumin suggests a difference of the globally unfolded state of the protein from the other species. The exchange mechanism of Trp60 of human alpha-lactalbumin is the low activation energy process in contrast with those of the bovine and goat proteins, although their global thermodynamic properties are similar to each other. Trp104 and Trp118 of alpha-lactalbumin exchange through the low activation energy process, and the reaction rates are affected by the local structural differences around the tryptophan residues among these proteins. The results presented in this paper indicate that the hydrogen exchange rate through the low activation energy process provides the information only about the local nature of a protein while that through the high activation energy process provides the information about the global nature of a protein.

Characterization of a partly folded protein by NMR methods: studies on the molten globule state of guinea pig alpha-lactalbumin.

NMR spectroscopy has been used to investigate the structure of a partially folded state of a protein, the molten globule or A-state of alpha-lactalbumin. The 1H NMR spectrum of this species differs substantially from those of both the native and fully unfolded states, reflecting the intermediate level of order. The resolution in the spectrum is limited by the widespread overlap and substantial line widths of many of the resonances. Methods have therefore been developed that exploit the well-resolved spectrum of the native protein to probe indirectly the A-state. A number of resonances of the A-state have been found to be substantially shifted from their positions in the spectrum of the unfolded state and have been identified through magnetization transfer with the native state, under conditions where the two states are interconverting. The most strongly perturbed residues in the A-state were found to be among those that form a hydrophobic core to the native structure. A number of amides were found to be highly protected from solvent exchange in the A-state. These have been identified through pH-jump experiments, which label them in the spectrum of the native protein. They were found to occur mainly in segments that are helical in the native structure. These results enable a model of the A-state to be proposed in which significant conformational freedom exists but where specific elements of native-like structure are preserved.

Structure and expression of the guinea-pig alpha-lactalbumin gene.

The entire guinea-pig alpha-lactalbumin gene was isolated from a genomic DNA library constructed in the bacteriophage lambda L47. The complete nucleotide sequence of the gene and its immediate 5' and 3' flanking sequences were determined and compared with those of the human and rat alpha-lactalbumin genes. This demonstrates that the size, organization and sequence of the exons is highly conserved between species, and reveals the presence of the highly conserved potential regulatory 'milk box' consensus sequence [RGAAGRAAA(N)TGGACAGAAATCAA(CG)TTTCTA] between positions -140 and -110 relative to the transcriptional start point. A guinea-pig LINE repeat sequence was also present in the 5' flanking region between positions -520 and -1195. Transfection of the alpha-lactalbumin gene cloned in a bovine papilloma virus vector into the mouse C127 and human MCF-7 mammary tumour cell-lines gave rise to stable but seemingly constitutive expression of alpha-lactalbumin. Expression was from the correct transcriptional start point, resulting in the accumulation of correctly processed mRNA and the secretion of alpha-lactalbumin into the culture medium.

Nuclear magnetic resonance characterization of aromatic residues of alpha-lactalbumins. Laser photo chemically induced dynamic nuclear polarization nuclear magnetic resonance studies of surface exposure.

The alpha-lactalbumins are involved as modifier proteins in lactose biosynthesis. Lactalbumins from different mammalian species are cross-reactive with the galactosyltransferases from others because of their homologous primary and tertiary structure. We have studied the surface exposure of several alpha-lactalbumins (bovine, goat, human, guinea pig, and rabbit) by the laser photo-CIDNP technique, an NMR method which measures the access of a photoexcited flavin dye to surface exposed Tyr, Trp, and His residues. An exposed histidine-68 residue exists in the bovine and goat species but is missing in other species and is replaced with a His-10 in the guinea pig species. The only exposed tryptophan residue was Trp-104, which cross-relaxed to nearby Trp-60. Cross-polarization to the latter residue was proven by taking free induction decays after extremely short light pulses (0.1 s). Furthermore, this cross-polarized Trp-60 resonance was absent in the guinea pig species, which has a substitution at position 60. The chemical shifts of these Trp residues were extremely similar in all species but rabbit, which has substituted His for a Tyr at position 103, situated close to both Trp-104 and -60. Three of the four tyrosines in bovine, goat, human, and guinea pig alpha-lactalbumin were exposed (the latter species has an extra Tyr in its sequence which was apparently not accessible). The rabbit species contains only two Tyr residues, the one which was not accessible (Tyr-50) being common to all species.

The characterization of the whey proteins of guinea-pig milk. The isolation and properties of alpha-lactalbumin.

1. The whey proteins of guinea-pig milk were examined by electrophoresis on paper, cellulose acetate, starch gel and polyacrylamide gel. 2. Two major proteins were detected, one of which was identified as blood serum albumin. 3. The major whey protein was isolated by CM-cellulose chromatography and on columns of Sephadex G-100. 4. The amino acid composition of the protein, taken in conjunction with its other properties, indicated that the major whey protein in guinea-pig milk is homologous with cow alpha-lactalbumin and that beta-lactoglobulin is absent from guinea-pig milk. 5. Guinea-pig alpha-lactalbumin, which was obtained crystalline, had mol.wt. 15800, N-terminal lysine and C-terminal glutamine.