Influence of protein and carbohydrate contents of soy protein hydrolysates on cell density and IgG production in animal cell cultures

The variety of compounds present in chemically defined media as well as media supplements makes it difficult to use a mechanistic approach to study the effect of supplement composition on culture functionality. Typical supplements, such as soy protein hydrolysates contain peptides, amino acids, carbohydrates, isoflavones, and saponins. To study the relative contribution of these compound classes, a set of hydrolysates were produced, containing 58‐83% proteinaceous material and 5‐21% carbohydrates. While the content of the different compounds classes varied, the composition (e.g., peptide profiles, carbohydrate composition) did not vary in hydrolysates. The hydrolysates were supplemented to a chemically defined medium in cell culture, based on equal weight and on equal protein levels. The latter showed that an increase in the carbohydrate concentration significantly (P value < 0.004) increased integral viable cell density (IVCD) (R = 0.7) and decreased total IgG (R = ?0.7) and specific IgG production (R = ?0.9). The extrapolation of effects of protein concentration showed that an increase in protein concentration increased total and specific IgG production and suppressed IVCD. In addition to proteins and carbohydrates, the functionality of soy protein hydrolysates may be modulated by the presence of other minor compounds. In the current study, the large differences in the balance between total proteins and total carbohydrates in the supplemented media seem to be a main factor influencing the balance between the viable cell density, total IgG, and specific IgG production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1396–1405, 2015     

Production of dehydroamino acid-containing peptides by Lactococcus lactis

Nisin is a pentacyclic peptide antibiotic produced by some Lactococcus lactis strains. Nisin contains dehydroresidues and thioether rings that are posttranslationally introduced by a membrane-associated enzyme complex, composed of a serine and threonine dehydratase NisB and the cyclase NisC. In addition, the transporter NisT is necessary for export of the modified peptide. We studied the potential of L. lactis expressing NisB and NisT to produce peptides whose serines and threonines are dehydrated. L. lactis containing the nisBT genes and a plasmid coding for a specific leader peptide fusion construct efficiently produced peptides with a series of non-naturally occurring multiple flanking dehydrobutyrines. We demonstrated NisB-mediated dehydration of serines and threonines in a C-terminal nisin(1-14) extension of nisin, which implies that also residues more distant from the leader peptide than those occurring in prenisin or any other lantibiotic can be modified. Furthermore, the feasibility and efficiency of generating a library of peptides containing dehydroresidues were demonstrated. In view of the particular shape and reactivity of dehydroamino acids, such a library provides a novel source for screening for peptides with desired biological and physicochemical properties.     

The active site of an algal prolyl 4-hydroxylase has a large structural plasticity

Prolyl 4-hydroxylases (P4Hs) are 2-oxoglutarate dioxygenases that catalyze the hydroxylation of peptidyl prolines. They play an important role in collagen synthesis, oxygen homeostasis, and plant cell wall formation. We describe four structures of a P4H from the green alga Chlamydomonas reinhardtii, two of the apoenzyme at 1.93 and 2.90 A resolution, one complexed with the competitive inhibitor Zn2+, and one with Zn2+ and pyridine 2,4-dicarboxylate (which is an analogue of 2-oxoglutarate) at 1.85 A resolution. The structures reveal the double-stranded beta-helix core fold (jellyroll motif), typical for 2-oxoglutarate dioxygenases. The catalytic site is at the center of an extended shallow groove lined by two flexible loops. Mutagenesis studies together with the crystallographic data indicate that this groove participates in the binding of the proline-rich peptide-substrates. It is discussed that the algal P4H and the catalytic domain of collagen P4Hs have notable structural similarities, suggesting that these enzymes form a separate structural subgroup of P4Hs different from the hypoxia-inducible factor P4Hs. Key structural differences between these two subgroups are described. These studies provide first insight into the structure-function relationships of the collagen P4Hs, which unlike the hypoxia-inducible factor P4Hs use proline-rich peptides as their substrates.     

High resolution crystal structures of unliganded and liganded human liver ACBP reveal a new mode of binding for the acyl-CoA ligand

The acyl-CoA binding protein (ACBP) is essential for the fatty acid metabolism, membrane structure, membrane fusion, and ceramide synthesis. Here high resolution crystal structures of human cytosolic liver ACBP, unliganded and liganded with a physiological ligand, myristoyl-CoA are described. The binding of the acyl-CoA molecule induces only few structural differences near the binding pocket. The crystal form of the liganded ACBP, which has two ACBP molecules in the asymmetric unit, shows that in human ACBP the same acyl-CoA binding pocket is present as previously described for the bovine and Plasmodium falciparum ACBP and the mode of binding of the 3'-phosphate-AMP moiety is conserved. Unexpectedly, in one of the acyl-CoA binding pockets the acyl moiety is bound in a reversed mode as compared with the bovine and P. falciparum structures. In this binding mode, the myristoyl-CoA molecule is fully ordered and bound across the two ACBP molecules of the crystallographic asymmetric unit: the 3'-phosphate-AMP moiety is bound in the binding pocket of one ACBP molecule and the acyl chain is bound in the pocket of the other ACBP molecule. The remaining binding pocket cavities of these two ACBP molecules are filled by other ligand fragments. This novel binding mode shows that the acyl moiety can flip out of its classical binding pocket and bind elsewhere, suggesting a mechanism for the acyl-CoA transfer between ACBP and the active site of a target enzyme. This mechanism is of possible relevance for the in vivo function of ACBP.     

Impact of QTL properties on the accuracy of multi-breed genomic prediction

Background

Although simulation studies show that combining multiple breeds in one reference population increases accuracy of genomic prediction, this is not always confirmed in empirical studies. This discrepancy might be due to the assumptions on quantitative trait loci (QTL) properties applied in simulation studies, including number of QTL, spectrum of QTL allele frequencies across breeds, and distribution of allele substitution effects. We investigated the effects of QTL properties and of including a random across- and within-breed animal effect in a genomic best linear unbiased prediction (GBLUP) model on accuracy of multi-breed genomic prediction using genotypes of Holstein-Friesian and Jersey cows.

Methods

Genotypes of three classes of variants obtained from whole-genome sequence data, with moderately low, very low or extremely low average minor allele frequencies (MAF), were imputed in 3000 Holstein-Friesian and 3000 Jersey cows that had real high-density genotypes. Phenotypes of traits controlled by QTL with different properties were simulated by sampling 100 or 1000 QTL from one class of variants and their allele substitution effects either randomly from a gamma distribution, or computed such that each QTL explained the same variance, i.e. rare alleles had a large effect. Genomic breeding values for 1000 selection candidates per breed were estimated using GBLUP modelsincluding a random across- and a within-breed animal effect.

Results

For all three classes of QTL allele frequency spectra, accuracies of genomic prediction were not affected by the addition of 2000 individuals of the other breed to a reference population of the same breed as the selection candidates. Accuracies of both single- and multi-breed genomic prediction decreased as MAF of QTL decreased, especially when rare alleles had a large effect. Accuracies of genomic prediction were similar for the models with and without a random within-breed animal effect, probably because of insufficient power to separate across- and within-breed animal effects.

Conclusions

Accuracy of both single- and multi-breed genomic prediction depends on the properties of the QTL that underlie the trait. As QTL MAF decreased, accuracy decreased, especially when rare alleles had a large effect. This demonstrates that QTL properties are key parameters that determine the accuracy of genomic prediction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0124-6) contains supplementary material, which is available to authorized users.     

Isolation and characterization of soluble protein from the green microalgae Tetraselmis sp

Extraction of high-value protein fractions for techno-functional applications in foods can considerably increase the commercial value of microalgae biomass. Proteins from Tetraselmis sp. were extracted and purified after cell disintegration by bead milling, centrifugation, ion exchange chromatography using the absorbent Streamline DEAE, and final decolorization by precipitation at pH 3.5. The algae soluble isolate was free from the intense color typical for algae products and contained 64% (w/w) proteins and 24% (w/w) carbohydrates. The final isolate showed solubility independent of ionic strength and 100% solubility at and above pH 5.5. Since most plant proteins used in foods show poor solubility in the pH range 5.5-6.5, the algae soluble protein isolate could be useful for techno-functional applications in this pH range.     

Structural biology of the thioester-dependent degradation and synthesis of fatty acids

The fatty acid degradation and synthesis pathways consist of the same four chemical transformations. These transformations are facilitated by conjugating the fatty acid, via a thioester bond, to coenzyme A or acyl carrier protein in, respectively, the degradation and synthesis pathways. These pathways are compartmentalized in the peroxisomes, mitochondria and cytosol of eukaryotic cells. Current structural knowledge of the enzymes comprising these pathways shows that the approximately 130 entries in the RCSB Protein Data Bank can be grouped into seven superfamilies. Multifunctional enzymes are important in both pathways.     

ICOS expression by activated human Th cells is enhanced by IL-12 and IL-23: increased ICOS expression enhances the effector function of both Th1 and Th2 cells

Previous mouse studies have shown that IL-4 increases the expression of ICOS on activated Th cells, resulting in enhanced ICOS expression on Th2 cells. In this study, we show that ICOS expression on human Th cells is not increased by IL-4, but by IL-12 and by IL-23 instead. Consequently, ICOS expression during IL-12-driven Th1 cell polarization was transiently increased compared with the levels on Th0 cells and IL-4-driven Th2 cells. Addition of IL-12 and/or IL-23 during restimulation increased ICOS expression to the same extent on pre-established Th1, Th2, and Th0 cells, indicating that ICOS levels are not stably imposed by prior polarization. In contrast to the findings in the mouse, IL-4 significantly suppressed the ICOS-enhancing effects of IL-12 and IL-23. The functional consequence of variable ICOS levels was shown in coculture experiments with cells expressing the ICOS-ligand B7-related protein 1 (either transfected Chinese hamster ovary cells or autologous dendritic cells). Ligation of ICOS on 2-day-preactivated effector cells increased their cytokine production to an extent proportional to their ICOS expression levels. As the ICOS-enhancing potentials of IL-12 and IL-23 were maintained for several days after stimulation, both on Th1 and Th2 cells, we propose the concept that local regulation of ICOS expression on activated Th cells by IL-12 and/or IL-23 may provide a powerful means to amplify effector T cell responses in peripheral tissues, independently of the polarized state of the Th cells.     

The planar conformation of a strained proline ring: a QM/MM study

QM and QM/MM energy calculations have been carried out on an atomic resolution structure of liganded triosephosphate isomerase (TIM) that has an active site proline (Pro168) in a planar conformation. The origin of the planarity of this proline has been identified. Steric interactions between the atoms of the proline ring and a tyrosine ring (Tyr166) on one side of the proline prevent the ring from adopting the up pucker (chi1 is approximately -30 degrees), while the side chain of a nearby alanine (Ala171) forbids the down pucker (chi1 is approximately +30 degrees). To obtain a proline conformation that is in agreement with the experimentally observed planar state, a quantum system of sufficient size is required and should at least include the nearby side chains of Tyr166, Ala171, and Glu129 to provide enough stabilization. It is argued that the current force fields for structure optimization do not describe strained protein fragments correctly. The proline is part of a catalytic loop that closes upon ligand binding. Comparison of the proline conformation in different TIM X-ray structures, indicates that in the closed conformation of TIM the proline is planar or nearly planar, while in the open conformation it is down puckered. This suggests that the planarity possibly plays a role in the overall catalytic cycle of TIM, presumable acting as a reservoir of energy that becomes available upon loop opening.     

Conductance and selectivity fluctuations in D127 mutants of the bacterial porin OmpF

A recent molecular dynamics study questioned the protonation state and physiological role of aspartate 127 (D127) of E. coli porin OmpF. To address that question we isolated two OmpF mutants with D127 either neutralized (D127N) or replaced by a positively charged lysine (D127K). The charge state of the residue at position 127 has clear effects on both conductance and selectivity. The D127K but not the D127N mutant expresses resilient conductance and selectivity fluctuations. These fluctuations reflect, we think, either changes in the ionization state of K127 and/or transitions between unstable subconformations as induced by the electrostatic repulsion between two positively charged residues, K127 and the nearby R167. Our results slightly favor the view that in WT OmpF residue D127 is deprotonated. As for the role of D127 in OmpF functionality, the gating of both mutants shows very similar sensitivity toward voltage as WT OmpF. Moreover, the current fluctuations of the D127K mutant were observed also in the absence of an applied electric field. We therefore dismiss D127 as a key residue in the control mechanism of the voltage-dependent gating of OmpF.