Metaproteomics reveals persistent and phylum-redundant metabolic functional stability in adult human gut microbiomes of Crohn's remission patients despite temporal variations in microbial taxa, genomes, and proteomes.

BACKGROUND: The gut microbiome plays a fundamental role in the human host's overall health by contributing key biological functions such as expanded metabolism and pathogen defense/immune control. In a healthy individual, the gut microbiome co-exists within the human host in a symbiotic, non-inflammatory relationship that enables mutual benefits, such as microbial degradation of indigestible food products into small molecules that the host can utilize, and enhanced pathogen defense. In abnormal conditions, such as Crohn's disease, this favorable metabolic relationship breaks down and a variety of undesirable activities result, including chronic inflammation and other health-related issues. It has been difficult, however, to elucidate the overall functional characteristics of this relationship because the microbiota can vary substantially in composition for healthy humans and possibly even more in individuals with gut disease conditions such as Crohn's disease. Overall, this suggests that microbial membership composition may not be the best way to characterize a phenotype. Alternatively, it seems to be more informative to examine and characterize the functional composition of a gut microbiome. Towards that end, this study examines 25 metaproteomes measured in several Crohn's disease patients' post-resection surgery across the course of 1 year, in order to examine persistence of microbial taxa, genes, proteins, and metabolic functional distributions across time in individuals whose microbiome might be more variable due to the gut disease condition. RESULTS: The measured metaproteomes were highly personalized, with all the temporally-related metaproteomes clustering most closely by individual. In general, the metaproteomes were remarkably distinct between individuals and to a lesser extent within individuals. This prompted a need to characterize the metaproteome at a higher functional level, which was achieved by annotating identified protein groups with KEGG orthologous groups to infer metabolic modules. At this level, similar and redundant metabolic functions across multiple phyla were observed across time and between individuals. Tracking through these various metabolic modules revealed a clear path from carbohydrate, lipid, and amino acid degradation to central metabolism and finally the production of fermentation products. CONCLUSIONS: The human gut metaproteome can vary quite substantially across time and individuals. However, despite substantial intra-individual variation in the metaproteomes, there is a clear persistence of conserved metabolic functions across time and individuals. Additionally, the persistence of these core functions is redundant across multiple phyla but is not always observable in the same sample. Finally, the gut microbiome's metabolism is not driven by a set of discrete linear pathways but a web of interconnected reactions facilitated by a network of enzymes that connect multiple molecules across multiple pathways.

Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NS0 cells.

Variable N-glycosylation at Asn(297) in the Fc region of recombinant therapeutic immunoglobulin G (IgG) molecules, specifically terminal galactosylation and sialylation, may affect both pharmacokinetic behavior and effector functions of recombinant therapeutic antibodies. We investigated the hypothesis that IgG Fc glycosylation can be controlled by manipulation of cellular nucleotide-sugar metabolism. In control cultures, N-glycans associated with the Fc domain of a recombinant humanized IgG1 produced by GS-NS0 cells in culture were predominantly biantennary, variably beta-galactosylated (average 0.3 mol galactose complex N-glycan(-1)) structures with no bisecting N-acetylglucosamine residues, sialylation, or alpha1,3-linked galactosylation evident. However, a variable proportion (5% to 15%) of high-mannose (Man5 to Man9) oligosaccharides were present. To manipulate the cellular content of the nucleotide sugar precursor required for galactosylation, UDP-Gal, we included either 10 mM glucosamine or 10 mM galactose in the culture medium. In the case of the former, a 17-fold increase in cellular UDP-N-acetylhexosamine content was observed, with a concomitant reduction (33%) in total UDP-hexose, although the ratio of UDP-Glc:UDP-Gal (4:1) was unchanged. Associated with these alterations in cellular UDP-sugar content was a significant reduction (57%) in the galactosylation of Fc-derived oligosaccharides. The proportion of high-mannose-type N-glycans (specifically Man5, the substrate for N-acetylglucosaminyltransferase I) at Asn(297) was unaffected. In contrast, inclusion of 10 mM galactose in culture specifically stimulated UDP-Gal content almost five-fold. However, this resulted in only a minimal, insignificant increase (6%) in beta1,4-galactosylation of Fc N-glycans. Sialylation was not improved upon the addition of the CMP-sialic acid (CMP-SA) precursor N-acetylmannosamine (20 mM), even with an associated 44-fold increase in cellular CMP-SA content. Analysis of recombinant IgG1 Fc glycosylation during batch culture showed that beta1,4-linked galactosylation declined slightly during culture, although, in the latter stages of culture, the release of proteases and glycosidases by lysed cells were likely to have contributed to the more dramatic drop in galactosylation. These data demonstrate: (i) the effect of steric hindrance on Fc N-glycan processing; (ii) the extent to which alterations in cellular nucleotide-sugar content may affect Fc N-glycan processing; and (iii) the potential for direct metabolic control of Fc N-glycosylation.

Determination of cytidine 5'-monophospho-N-acetylneuraminic acid pool size in cell culture scale using high-performance anion-exchange chromatography with pulsed amperometric detection.

A simultaneous determination of cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) and its metabolic products, cytidine, CMP and Neu5Ac by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using a Carbo-Pac PA1 column is described. Preparation of the samples involved a single purification step of the crude cell extract on DEAE-Sepharose. The method is adequate to quantify the amount of CMP-Neu5Ac produced by one culture dish; equivalent to 6.10(6) cells. In addition, a method for desalting and recovery of the separated material was developed to determine the cellular concentration of CMP-Neu5Ac in Madin Darby canine kidney (MDCK) cells. The addition of 5 mM N-acetylmannosamine to the culture medium gave rise to a 6.4-fold elevation of this value.

Assay of sialyltransferase activity by reversed-phase ion-pair high-performance liquid chromatography.

Sialyltransferases (CMP-N-acetylneuraminic acid:glycoprotein sialyltransferases, EC 2.4.99.1) are involved in the transfer of a sialic acid moiety from CMP-N-acetylneuraminic acid (CMP-NeuAc) to an oligosaccharide side-chain of an acceptor, asialoglycoprotein (AGP), according to the following reaction: CMP-NeuAc + AGP----NeuAc-O-AGP + CMP. This enzyme occurs in elevated levels in the sera of patients with a wide variety of neoplastic diseases and its assay might be useful in monitoring treatment. Radioactive CMP-NeuAc has been used in assays and the radioactive sialylated product separated and counted by liquid scintillation spectrometry. This study shows that a simple, rapid, non-radiochemically based high-performance liquid chromatographic method developed for the analysis of CMP-sialic acid synthetase can be used for the quantitation of sialyltransferase activity by monitoring simultaneously the utilization of CMP-NeuAc and the release of CMP. We describe the application of this method to assay of commercially available sialyltransferase activity and to activities from synovial, ascites and gastric fluids.

Glycolipid sialosyltransferase activity in synaptosomes exhibits a product specificity for (2-8)disialosyl lactosyl ceramide (ganglioside GD3).

Intact synaptosomes prepared from 28-day-old rat brains were incubated with CMP-N-acetyl-(14C) neuraminic acid in Krebs-Henseleit buffer in an atmosphere of 95% O2: 5% CO2, at 37 degrees C. The activity of CMP-NANA:ganglioside sialosyltransferase using endogenous acceptors was 0.84 pmoles NANA transferred/mg synaptosomal protein/hr. Analysis of the distribution of labeled sialic acid revealed that GD3 ganglioside (alpha 2----8 disialosyl, alpha 2----3 galactosyl, beta 1----4 glucosyl, beta 1----1-ceramide) was the major product in the membrane carrying 32% of the total lipid bound label. Treatment of the reaction products with Clostridium neuraminidase liberated labeled sialic acid from GD3 and yielded labeled GM3, then unlabeled lactosyl ceramide. Lac-cer and GM3 are present in small amounts in synaptosomes, and GD3 represents less than 2% of the total ganglioside. Our findings indicate that the sialosyltransferase activity of synaptosomes exhibits a preferential product specificity for the small pool of synaptosomal membrane GD3 ganglioside that may be formed in situ, via sialosylation of its precursor (GM3 or lactosyl ceramide) which pre-exists in the synaptosomal plasma membrane. The second major labeled product quantitatively was GD1a whose precursor substrate, GM1, is quite abundant in the membrane, so that the conversion rate of GM1 to GD1a was low in comparison with GD3 formation. Sialosylation of other synaptosomal membrane gangliosides was negligible.