Activation of the G-protein coupled receptor GPR35 by human milk oligosaccharides through different pathways

Abstract Numerous benefits of breastfeeding over infant formula are fully established. The superiority of human milk over bovine milk-based formula is partly due to human milk oligosaccharides (HMOs), a family of over 100 molecules present specifically and substantially in human milk that resemble mucosal glycans. To uncover novel physiological functions and pathways of HMOs, we screened a panel of 165 G-protein coupled receptors (GPCRs) using a blend of 6 HMOs (3′-O-sialyllactose (3′SL), 6′-O-sialyllactose (6′SL), lacto-N-tetraose (LNT), lacto-N-neo-tetraose (LNnT), 2-O-fucosyllactose (2′FL), and difucosyllactose (diFL)), and followed up positive hits with standard receptor assays. The HMO blend specifically activated GPR35. LNT and 6′SL individually activated GPR35, and they showed synergy when used together. In addition, in vitro fermentation of infant stool samples showed that 2′FL upregulates the production of the GPR35 agonist kynurenic acid (KYNA) by the microbiota. LNT + 6′SL and KYNA showed additive activation of GPR35. Activation by 6′SL and LNT of GPR35, a receptor mediating attenuation of pain and colitis, is to our knowledge the first demonstration of GPCR activation by any HMO. In addition, we demonstrated a remarkable cooperation between nutrition and microbiota towards activation of a host receptor highlighting the close interplay between environment and host-microbe interactions.

Download Full-text

Akkermansia muciniphila uses human milk oligosaccharides to thrive in the early life conditions in vitro

Scientific Reports ◽

10.1038/s41598-020-71113-8 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Ioannis Kostopoulos ◽

Janneke Elzinga ◽

Noora Ottman ◽

Jay T. Klievink ◽

Bernadet Blijenberg ◽

...

Keyword(s):

Human Milk ◽

Early Life ◽

Human Milk Oligosaccharides ◽

Milk Oligosaccharides ◽

Akkermansia Muciniphila ◽

Life Conditions ◽

Early Life Conditions

Download Full-text

Human milk oligosaccharides reduceEntamoeba histolyticaattachment and cytotoxicityin vitro

British Journal Of Nutrition ◽

10.1017/s0007114511007392 ◽

2012 ◽

Vol 108 (10) ◽

pp. 1839-1846 ◽

Cited By ~ 69

Author(s):

Evelyn Jantscher-Krenn ◽

Tineke Lauwaet ◽

Laura A. Bliss ◽

Sharon L. Reed ◽

Frances D. Gillin ◽

...

Keyword(s):

Human Milk ◽

Protozoan Parasite ◽

Bacterial Attachment ◽

Dependent Manner ◽

Human Milk Oligosaccharides ◽

Milk Oligosaccharides ◽

Beneficial Effects ◽

Ht 29

Human milk oligosaccharides (HMO), complex sugars that are highly abundant in breast milk, block viral and bacterial attachment to the infant's intestinal epithelium and lower the risk of infections. We hypothesised that HMO also prevent infections with the protozoan parasiteEntamoeba histolytica,as its major virulence factor is a lectin that facilitates parasite attachment and cytotoxicity and binds galactose (Gal) andN-acetyl-galactosamine. HMO contain Gal, are only minimally digested in the small intestine and reach the colon, the site ofE. histolyticainfection. The objective of the present study was to investigate whether HMO reduceE. histolyticaattachment and cytotoxicity. Ourin vitroresults show that physiological concentrations of isolated, pooled HMO detachE. histolyticaby more than 80 %. In addition, HMO rescueE. histolytica-induced destruction of human intestinal epithelial HT-29 cells in a dose-dependent manner. The cytoprotective effects were structure-specific. Lacto-N-tetraose with its terminal Gal rescued up to 80 % of the HT-29 cells, while HMO with fucose α1–2-linked to the terminal Gal had no effect. Galacto-oligosaccharides (GOS), which also contain terminal Gal and are currently added to infant formula to mimic some of the beneficial effects of HMO, completely abolishedE. histolyticaattachment and cytotoxicity at 8 mg/ml. Although our results need to be confirmedin vivo, they may provide one explanation for why breast-fed infants are at lower risk ofE. histolyticainfections. HMO and GOS are heat tolerant, stable, safe and in the case of GOS, inexpensive, which could make them valuable candidates as alternative preventive and therapeutic anti-amoebic agents.

Download Full-text

Human Milk Oligosaccharides Protect Against Enteropathogenic Escherichia coli Attachment In Vitro and EPEC Colonization in Suckling Mice

Journal of Pediatric Gastroenterology and Nutrition ◽

10.1097/mpg.0000000000000172 ◽

2014 ◽

Vol 58 (2) ◽

pp. 165-168 ◽

Cited By ~ 49

Author(s):

Carolin F. Manthey ◽

Chloe A. Autran ◽

Lars Eckmann ◽

Lars Bode

Keyword(s):

Escherichia Coli ◽

Human Milk ◽

Enteropathogenic Escherichia Coli ◽

Human Milk Oligosaccharides ◽

Milk Oligosaccharides ◽

Suckling Mice

Download Full-text

Microbial Composition and In Vitro Fermentation Patterns of Human Milk Oligosaccharides and Prebiotics Differ between Formula-Fed and Sow-Reared Piglets

Journal of Nutrition ◽

10.3945/jn.111.154427 ◽

2012 ◽

Vol 142 (4) ◽

pp. 681-689 ◽

Cited By ~ 58

Author(s):

Min Li ◽

Laura L. Bauer ◽

Xin Chen ◽

Mei Wang ◽

Theresa B. Kuhlenschmidt ◽

...

Keyword(s):

Human Milk ◽

Human Milk Oligosaccharides ◽

Microbial Composition ◽

Milk Oligosaccharides ◽

In Vitro Fermentation

Download Full-text

Human Milk Oligosaccharides in the Prevention of Necrotizing Enterocolitis: A Journey From in vitro and in vivo Models to Mother-Infant Cohort Studies

Frontiers in Pediatrics ◽

10.3389/fped.2018.00385 ◽

2018 ◽

Vol 6 ◽

Cited By ~ 25

Author(s):

Lars Bode

Keyword(s):

Human Milk ◽

Cohort Studies ◽

Necrotizing Enterocolitis ◽

Human Milk Oligosaccharides ◽

Milk Oligosaccharides ◽

In Vivo Models

Download Full-text

G Protein-coupled Receptor Kinase 2–mediated Phosphorylation of Ezrin Is Required for G Protein-coupled Receptor–dependent Reorganization of the Actin Cytoskeleton

Molecular Biology of the Cell ◽

10.1091/mbc.e04-10-0877 ◽

2005 ◽

Vol 16 (7) ◽

pp. 3088-3099 ◽

Cited By ~ 100

Author(s):

Sarah H. Cant ◽

Julie A. Pitcher

Keyword(s):

G Protein ◽

Phosphorylation Site ◽

Rho Kinase ◽

Receptor Kinase ◽

G Protein Coupled Receptor ◽

Glutathione S Transferase ◽

Membrane Ruffling ◽

Gpcr Activation ◽

G Protein Coupled

G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes activated G protein-coupled receptors (GPCRs). Here, we identify ezrin as a novel non-GPCR substrate of GRK2. GRK2 phosphorylates glutathione S-transferase (GST)-ezrin, but not an ezrin fusion protein lacking threonine 567 (T567), in vitro. These results suggest that T567, the regulatory phosphorylation site responsible for maintaining ezrin in its active conformation, represents the principle site of GRK2-mediated phosphorylation. Two lines of evidence indicate that GRK2-mediated ezrin-radixinmoesin (ERM) phosphorylation serves to link GPCR activation to cytoskeletal reorganization. First, in Hep2 cells muscarinic M1 receptor (M1MR) activation causes membrane ruffling. This ruffling response is ERM dependent and is accompanied by ERM phosphorylation. Inhibition of GRK2, but not rho kinase or protein kinase C, prevents ERM phosphorylation and membrane ruffling. Second, agonist-induced internalization of the β2-adrenergic receptor (β2AR) and M1MR is accompanied by ERM phosphorylation and localization of phosphorylated ERM to receptor-containing endocytic vesicles. The colocalization of internalized β2AR and phosphorylated ERM is not dependent on Na+/H+ exchanger regulatory factor binding to the β2AR. Inhibition of ezrin function impedes β2AR internalization, further linking GPCR activation, GRK activity, and ezrin function. Overall, our results suggest that GRK2 serves not only to attenuate but also to transduce GPCR-mediated signals.

Download Full-text

Assessment of the synbiotic properites of human milk oligosaccharides and Bifidobacterium longum subsp. infantis in vitro and in humanised mice

Beneficial Microbes ◽

10.3920/bm2016.0138 ◽

2017 ◽

Vol 8 (2) ◽

pp. 281-289 ◽

Cited By ~ 4

Author(s):

S. Musilova ◽

N. Modrackova ◽

P. Hermanova ◽

T. Hudcovic ◽

R. Svejstil ◽

...

Keyword(s):

Caesarean Section ◽

Human Milk ◽

Pathogenic Bacteria ◽

Bifidobacterium Longum ◽

Mode Of Delivery ◽

Human Milk Oligosaccharides ◽

Milk Oligosaccharides ◽

High Concentrations

The mode of delivery plays a crucial role in infant gastrointestinal tract colonisation, which in the case of caesarean section is characterised by the presence of clostridia and low bifidobacterial counts. Gut colonisation can be modified by probiotics, prebiotics or synbiotics. Human milk oligosaccharides (HMOs) are infant prebiotics that show a bifidogenic effect. Moreover, genome sequencing of Bifidobacterium longum subsp. infantis within the infant microbiome revealed adaptations for milk utilisation. This study aimed to evaluate the synbiotic effect of B. longum subsp. infantis, HMOs and human milk (HM) both in vitro and in vivo (in a humanised mouse model) in the presence of faecal microbiota from infants born by caesarean section. The combination of B. longum and HMOs or HM reduced the clostridia and G-bacteria counts both in vitro and in vivo. The bifidobacterial population in vitro significantly increased and produce high concentrations of acetate and lactate. In vitro competition assays confirmed that the tested bifidobacterial strain is a potential probiotic for infants and, together with HMOs or HM, acts as a synbiotic. It is also able to inhibit potentially pathogenic bacteria. The synbiotic effects identified in vitro were not observed in vivo. However, there was a significant reduction in clostridia counts in both experimental animal groups (HMOs + B. longum and HM + B. longum), and a specific immune response via increased interleukin (IL)-10 and IL-6 production. Animal models do not perfectly mimic human conditions; however, they are essential for testing the safety of functional foods.

Download Full-text

Oligosaccharides Released from Milk Glycoproteins Are Selective Growth Substrates for Infant-Associated Bifidobacteria

Applied and Environmental Microbiology ◽

10.1128/aem.00547-16 ◽

2016 ◽

Vol 82 (12) ◽

pp. 3622-3630 ◽

Cited By ~ 67

Author(s):

Sercan Karav ◽

Annabelle Le Parc ◽

Juliana Maria Leite Nobrega de Moura Bell ◽

Steven A. Frese ◽

Nina Kirmiz ◽

...

Keyword(s):

Mass Spectrometry ◽

Human Milk ◽

Bifidobacterium Longum ◽

Bovine Milk ◽

Selective Growth ◽

Whey Protein Concentrate ◽

Human Milk Oligosaccharides ◽

Milk Oligosaccharides ◽

Content Type ◽

Growth Substrates

ABSTRACTMilk, in addition to nourishing the neonate, provides a range of complex glycans whose construction ensures a specific enrichment of key members of the gut microbiota in the nursing infant, a consortium known as the milk-oriented microbiome. Milk glycoproteins are thought to function similarly, as specific growth substrates for bifidobacteria common to the breast-fed infant gut. Recently, a cell wall-associated endo-β-N-acetylglucosaminidase (EndoBI-1) found in various infant-borne bifidobacteria was shown to remove a range of intactN-linked glycans. We hypothesized that these released oligosaccharide structures can serve as a sole source for the selective growth of bifidobacteria. We demonstrated that EndoBI-1 releasedN-glycans from concentrated bovine colostrum at the pilot scale. EndoBI-1-releasedN-glycans supported the rapid growth ofBifidobacterium longumsubsp.infantis(B. infantis), a species that grows well on human milk oligosaccharides, but did not support growth ofBifidobacterium animalissubsp.lactis(B. lactis), a species which does not. Conversely,B. infantisATCC 15697 did not grow on the deglycosylated milk protein fraction, clearly demonstrating that the glycan portion of milk glycoproteins provided the key substrate for growth. Mass spectrometry-based profiling revealed thatB. infantisconsumed 73% of neutral and 92% of sialylatedN-glycans, whileB. lactisdegraded only 11% of neutral and virtually no (<1%) sialylatedN-glycans. These results provide mechanistic support thatN-linked glycoproteins from milk serve as selective substrates for the enrichment of infant-associated bifidobacteria capable of carrying out the initial deglycosylation. Moreover, releasedN-glycans were better growth substrates than the intact milk glycoproteins, suggesting that EndoBI-1 cleavage is a key initial step in consumption of glycoproteins. Finally, the variety ofN-glycans released from bovine milk glycoproteins suggests that they may serve as novel prebiotic substrates with selective properties similar to those of human milk oligosaccharides.IMPORTANCEIt has been previously shown that glycoproteins serve as growth substrates for bifidobacteria. However, which part of a glycoprotein (glycans or polypeptides) is responsible for this function was not known. In this study, we used a novel enzyme to cleave conjugatedN-glycans from milk glycoproteins and tested their consumption by various bifidobacteria. The results showed that the glycans selectively stimulated the growth ofB. infantis, which is a key infant gut microbe. The selectivity of consumption of individualN-glycans was determined using advanced mass spectrometry (nano-liquid chromatography chip–quadrupole time of flight mass spectrometry [nano-LC-Chip-Q-TOF MS]) to reveal thatB. infantiscan consume the range of glycan structures released from whey protein concentrate.

Download Full-text