39 research outputs found
Rosiglitazone Induces Mitochondrial Biogenesis in Differentiated Murine 3T3-L1 and C3H/10T1/2 Adipocytes
Growing evidence indicates that PPARγ agonists, including rosiglitazone (RSG), induce adipose mitochondrial biogenesis. By systematically analyzing mitochondrial gene expression in two common murine adipocyte models, the current study aimed to further establish the direct role of RSG and capture temporal changes in gene transcription. Microarray profiling revealed that in fully differentiated 3T3-L1 and C3H/10T1/2 adipocytes treated with RSG or DMSO vehicle for 1, 2, 4, 7, 24, and 48 hrs, RSG overwhelmingly increased mitochondrial gene transcripts time dependently. The timing of the increases was consistent with the cascade of organelle biogenesis, that is, initiated by induction of transcription factor(s), followed by increases in the biosynthesis machinery, and then by increases in functional components. The transcriptional increases were further validated by increased mitochondrial staining, citrate synthase activity, and O2 consumption, and were found to be associated with increased adiponectin secretion. The work provided further insight on the mechanism of PPARγ-induced mitochondrial biogenesis in differentiated adipocytes
Site-Specific Glycosylation of Secretory Immunoglobulin A from Human Colostrum
Secretory
immunoglobulin A (sIgA) is a major glycoprotein in milk and plays
a key role in mediating immune protection of the gut mucosa. Although
it is a highly glycosylated protein, its site-specific glycosylation
and associated glycan micro-heterogeneity have still not been fully
elucidated. In this study, the site-specific glycosylation of sIgA
isolated from human colostrum (<i>n</i> = 3) was analyzed
using a combination of LC–MS and LC–MS/MS and in-house
software (Glycopeptide Finder). The majority of the glycans found
are biantennary structures with one or more acidic Neu5Ac residues;
however, a large fraction belonged to truncated complex structures
with terminal GlcNAc. Multiple glycosites were identified with nearly
30 glycan compositions located at seven sites on the secretory component,
six compositions at a single site on the J chain, and 16 compositions
at five sites on the IgA heavy (H) chain. Site-specific heterogeneity
and relative quantitation of each composition and the extent of occupation
at each site were determined using nonspecific proteases. Additionally,
54 O-linked glycan compositions located at the IgA1 hinge region (HR)
were identified by comparison against a theoretical O-glycopeptide
library. This represents the most comprehensive report to date detailing
the complexity of glycan micro-heterogeneity with relative quantitation
of glycoforms for each glycosylation site on milk sIgA. This strategy
further provides a general method for determining site-specific glycosylation
in large protein complexes
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Human milk glycomics and gut microbial genomics in infant feces show a correlation between human milk oligosaccharides and gut microbiota: a proof-of-concept study.
Human milk oligosaccharides (HMOs) play a key role in shaping and maintaining a healthy infant gut microbiota. This article demonstrates the potential of combining recent advances in glycomics and genomics to correlate abundances of fecal microbes and fecal HMOs. Serial fecal specimens from two healthy breast-fed infants were analyzed by bacterial DNA sequencing to characterize the microbiota and by mass spectrometry to determine abundances of specific HMOs that passed through the intestinal tract without being consumed by the luminal bacteria. In both infants, the fecal bacterial population shifted from non-HMO-consuming microbes to HMO-consuming bacteria during the first few weeks of life. An initial rise in fecal HMOs corresponded with bacterial populations composed primarily of non-HMO-consuming Enterobacteriaceae and Staphylococcaeae. This was followed by decreases in fecal HMOs as the proportion of HMO-consuming Bacteroidaceae and Bifidobacteriaceae increased. Analysis of HMO structures with isomer differentiation revealed that HMO consumption is highly structure-specific, with unique isomers being consumed and others passing through the gut unaltered. These results represent a proof-of-concept and are consistent with the highly selective, prebiotic effect of HMOs in shaping the gut microbiota in the first weeks of life. The analysis of selective fecal bacterial substrates as a measure of alterations in the gut microbiota may be a potential marker of dysbiosis
In-Gel Nonspecific Proteolysis for Elucidating Glycoproteins: A Method for Targeted Protein-Specific Glycosylation Analysis in Complex Protein Mixtures
Determining protein-specific glycosylation in protein
mixtures
remains a difficult task. A common approach is to use gel electrophoresis
to isolate the protein followed by glycan release from the identified
band. However, gel bands are often composed of several proteins. Hence,
release of glycans from specific bands often yields products not from
a single protein but a composite. As an alternative, we present an
approach whereby glycans are released with peptide tags allowing verification
of glycans bound to specific proteins. We term the process in-gel
nonspecific proteolysis for elucidating glycoproteins (INPEG). INPEG
combines rapid gel separation of a protein mixture with in-gel nonspecific
proteolysis of protein bands followed by tandem mass spectrometry
(MS) analysis of the resulting N- and O-glycopeptides. Here, in-gel
digestion is shown for the first time with nonspecific and broad specific
proteases such as Pronase, proteinase K, pepsin, papain, and subtilisin.
Tandem MS analysis of the resulting glycopeptides separated on a porous
graphitized carbon (PGC) chip was achieved via nanoflow liquid chromatography
coupled with quadrupole time-of-flight mass spectrometry (nano-LC/Q-TOF
MS). In this study, rapid and automated glycopeptide assignment was
achieved via an in-house software (Glycopeptide Finder) based on a
combination of accurate mass measurement, tandem MS data, and predetermined
protein identification (obtained via routine shotgun analysis). INPEG
is here initially validated for O-glycosylation (κ casein) and
N-glycosylation (ribonuclease B). Applications of INPEG were further
demonstrated for the rapid determination of detailed site-specific
glycosylation of lactoferrin and transferrin following gel separation
and INPEG analysis on crude bovine milk and human serum, respectively
Human Milk Glycomics and Gut Microbial Genomics in Infant Feces Show a Correlation between Human Milk Oligosaccharides and Gut Microbiota: A Proof-of-Concept Study
Human
milk oligosaccharides (HMOs) play a key role in shaping and
maintaining a healthy infant gut microbiota. This article demonstrates
the potential of combining recent advances in glycomics and genomics
to correlate abundances of fecal microbes and fecal HMOs. Serial fecal
specimens from two healthy breast-fed infants were analyzed by bacterial
DNA sequencing to characterize the microbiota and by mass spectrometry
to determine abundances of specific HMOs that passed through the intestinal
tract without being consumed by the luminal bacteria. In both infants,
the fecal bacterial population shifted from non-HMO-consuming microbes
to HMO-consuming bacteria during the first few weeks of life. An initial
rise in fecal HMOs corresponded with bacterial populations composed
primarily of non-HMO-consuming Enterobacteriaceae and Staphylococcaeae. This was followed
by decreases in fecal HMOs as the proportion of HMO-consuming Bacteroidaceae and Bifidobacteriaceae increased. Analysis of HMO structures with isomer differentiation
revealed that HMO consumption is highly structure-specific, with unique
isomers being consumed and others passing through the gut unaltered.
These results represent a proof-of-concept and are consistent with
the highly selective, prebiotic effect of HMOs in shaping the gut
microbiota in the first weeks of life. The analysis of selective fecal
bacterial substrates as a measure of alterations in the gut microbiota
may be a potential marker of dysbiosis
Automated Assignments of N- and O‑Site Specific Glycosylation with Extensive Glycan Heterogeneity of Glycoprotein Mixtures
Site-specific glycosylation (SSG)
of glycoproteins remains a considerable
challenge and limits further progress in the areas of proteomics and
glycomics. Effective methods require new approaches in sample preparation,
detection, and data analysis. While the field has advanced in sample
preparation and detection, automated data analysis remains an important
goal. A new bioinformatics approach implemented in software called
GP Finder automatically distinguishes correct assignments from random
matches and complements experimental techniques that are optimal for
glycopeptides, including nonspecific proteolysis and high mass resolution
liquid chromatography/tandem mass spectrometry (LC/MS/MS). SSG for
multiple N- and O-glycosylation sites, including extensive glycan
heterogeneity, was annotated for single proteins and protein mixtures
with a 5% false-discovery rate, generating hundreds of nonrandom glycopeptide
matches and demonstrating the proof-of-concept for a self-consistency
scoring algorithm shown to be compliant with the target-decoy approach
(TDA). The approach was further applied to a mixture of N-glycoproteins
from unprocessed human milk and O-glycoproteins from very-low-density-lipoprotein
(vLDL) particles