Biochemical Foundations of Health and Energy Conservation in Hibernating Free-ranging Subadult Brown Bear Ursus arctos.

Brown bears (Ursus arctos) hibernate for 5-7 months without eating, drinking, urinating, and defecating at a metabolic rate of only 25% of the summer activity rate. Nonetheless, they emerge healthy and alert in spring. We quantified the biochemical adaptations for hibernation by comparing the proteome, metabolome, and hematological features of blood from hibernating and active free-ranging subadult brown bears with a focus on conservation of health and energy. We found that total plasma protein concentration increased during hibernation, even though the concentrations of most individual plasma proteins decreased, as did the white blood cell types. Strikingly, antimicrobial defense proteins increased in concentration. Central functions in hibernation involving the coagulation response and protease inhibition, as well as lipid transport and metabolism, were upheld by increased levels of very few key or broad specificity proteins. The changes in coagulation factor levels matched the changes in activity measurements. A dramatic 45-fold increase in sex hormone-binding globulin levels during hibernation draws, for the first time, attention to its significant but unknown role in maintaining hibernation physiology. We propose that energy for the costly protein synthesis is reduced by three mechanisms as follows: (i) dehydration, which increases protein concentration without de novo synthesis; (ii) reduced protein degradation rates due to a 6 °C reduction in body temperature and decreased protease activity; and (iii) a marked redistribution of energy resources only increasing de novo synthesis of a few key proteins. The comprehensive global data identified novel biochemical strategies for bear adaptations to the extreme condition of hibernation and have implications for our understanding of physiology in general.

Glycosylations and truncations of functional cereal phytases expressed and secreted by Pichia pastoris documented by mass spectrometry.

Cereal purple acid phosphatase-type phytases, PAPhy, play an essential role in making phosphate accessible to mammalian digestion and reducing the environmental impact of manure. Studying the potential of PAPhy requires easy access to the enzymes. For that purpose wheat and barley isophytases have been expressed in Pichia pastoris from constructs encoding the alpha-mating factor at the N-termini and a His6 tag before the stop codon in all constructs. A protein chemical study of a C-terminally truncated recombinant wheat phytase, r-TaPAPhy_b2, was carried out to clarifying the posttranslational processing of proteins secreted from P. pastoris. Extensive mass spectrometric sequencing of tryptic, chymotryptic and AspN derived peptides of both the native and endoH deglycosylated forms showed: (i) All mating factor derived sequence had been removed and further unspecific proteolysis left highly heterogeneous N-terminal variant forms of r-TaPAPhy; (ii) The His6 tag had been retained or slightly truncated; (iii) All seven potential N-glycan sites were glycosylated except for two sites which were partially glycosylated by ca. 90% and 30%; (iv) Among the nine cysteine residues of this phytase, the most N-terminal residue is free, whereas the remaining eight appear to be disulfide bonded. It is noteworthy that already the first step in ESI-MS/MS sequencing had fragmented the hyper glycosylated peptides into free Z, Y and X mass spectrometric glycan fragments attached to the peptide.

Extensive post-translational processing of potato tuber storage proteins and vacuolar targeting.

Potato tuber storage proteins were obtained from vacuoles isolated from field-grown starch potato tubers cv. Kuras. Vacuole sap proteins fractionated by gel filtration were studied by mass spectrometric analyses of trypsin and chymotrypsin digestions. The tuber vacuole appears to be a typical protein storage vacuole absent of proteolytic and glycolytic enzymes. The major soluble storage proteins included 28 Kunitz protease inhibitors, nine protease inhibitors 1, eight protease inhibitors 2, two carboxypeptidase inhibitors, eight patatins and five lipoxygenases (lox), which all showed cultivar-specific sequence variations. These proteins, except for lox, have typical endoplasmic reticulum (ER) signal peptides and putative vacuolar sorting determinants of either the sequence or structure specific type or the C-terminal type, or both. Unexpectedly, sap protein variants imported via the ER showed multiple molecular forms because of extensive and unspecific proteolytic cleavage of exposed N- and C-terminal propeptides and surface loops, in spite of the abundance of protease inhibitors. Some propeptides are potential novel vacuolar targeting peptides. In the insoluble vacuole fraction two variants of phytepsin (aspartate protease) were identified. These are most probably the processing enzymes of potato tuber vacuolar proteins. Database Proteome data have been submitted to the PRIDE database under accession number 17707.

Different site-specific N-glycan types in wheat (Triticum aestivum L.) PAP phytase.

Phytase activity in grain is essential to make phosphate available to cell metabolism, and in food and feed. Cereals contain the purple acid phosphatase type of phytases (PAPhy). Mature wheat grain is dominated by TaPAPhy_a which, in the present work, has been characterized by extensive peptide and glycopeptide sequencing by mass spectrometry. Seven N-linked glycosylation sites were found. Three of these sites were dominated by variant forms of the XylMan(3)FucGlcNAc(2), i.e. the HRP-type of glycan. Complex-type glycans with one or two additional GlcNAc were observed, however in trace amounts only. At four sites the glycan consisted of a single GlcNAc residue. The mature protein is ca. 500 residues in size and appears to be truncated at the N- and C-termini.