Elements of the Endomucin Extracellular Domain Essential for VEGF-Induced VEGFR2 Activity.

Endomucin (EMCN) is the type I transmembrane glycoprotein, mucin-like component of the endothelial cell glycocalyx. We have previously shown that EMCN is necessary for vascular endothelial growth factor (VEGF)-induced VEGF receptor 2 (VEGFR2) internalization and downstream signaling. To explore the structural components of EMCN that are necessary for its function and the molecular mechanism of EMCN in VEGF-induced endothelial functions, we generated a series of mouse EMCN truncation mutants and examined their ability to rescue VEGF-induced endothelial functions in human primary endothelial cells (EC) in which endogenous EMCN had been knocked down using siRNA. Expression of the mouse full-length EMCN (FL EMCN) and the extracellular domain truncation mutants ∆21-81 EMCN and ∆21-121 EMCN, but not the shortest mutant ∆21-161 EMCN, successfully rescued the VEGF-induced EC migration, tube formation, and proliferation. ∆21-161 EMCN failed to interact with VEGFR2 and did not facilitate VEGFR2 internalization. Deletion of COSMC (C1GalT1C1) revealed that the abundant mucin-type O-glycans were not required for its VEGFR2-related functions. Mutation of the two N-glycosylation sites on ∆21-121 EMCN abolished its interaction with VEGFR2 and its function in VEGFR2 internalization. These results reveal ∆21-121 EMCN as the minimal extracellular domain sufficient for VEGFR2-mediated endothelial function and demonstrate an important role for N-glycosylation in VEGFR2 interaction, internalization, and angiogenic activity.

Characterization of a sialate-O-acetylesterase (NanS) from the oral pathogen Tannerella forsythia that enhances sialic acid release by NanH, its cognate sialidase.

Tannerella forsythia, a Gram-negative member of the Bacteroidetes has evolved to harvest and utilize sialic acid. The most common sialic acid in humans is a mono-N-acetylated version termed Neu5Ac (5-N-acetyl-neuraminic acid). Many bacteria are known to access sialic acid using sialidase enzymes. However, in humans a high proportion of sialic acid contains a second acetyl group attached via an O-group, i.e. chiefly O-acetylated Neu5,9Ac2 or Neu5,4Ac2. This diacetylated sialic acid is not cleaved efficiently by many sialidases and in order to access diacetylated sialic acid, some organisms produce sialate-O-acetylesterases that catalyse the removal of the second acetyl group. In the present study, we performed bioinformatic and biochemical characterization of a putative sialate-O-acetylesterase from T. forsythia (NanS), which contains two putative SGNH-hydrolase domains related to sialate-O-acetylesterases from a range of organisms. Purification of recombinant NanS revealed an esterase that has activity against Neu5,9Ac2 and its glycolyl form Neu5Gc,9Ac. Importantly, the enzyme did not remove acetyl groups positioned at the 4-O position (Neu5,4Ac2). In addition NanS can act upon complex N-glycans released from a glycoprotein [erythropoietin (EPO)], bovine submaxillary mucin and oral epithelial cell-bound glycans. When incubated with its cognate sialidase, NanS increased sialic acid release from mucin and oral epithelial cell surfaces, implying that this esterase improves sialic acid harvesting for this pathogen and potentially other members of the oral microbiome. In summary, we have characterized a novel sialate-O-acetylesterase that contributes to the sialobiology of this important human pathogen and has potential applications in the analysis of sialic acid diacetylation of biologics in the pharmaceutical industry.

Histochemical characterization of the mucins of the alimentary tract of the grass snake, Natrix natrix (Colubridae).

Characterization of mucins in the alimentary tract of the grass snake, Natrix natrix was performed by histochemical (PAS, Alcian Blue, pH 2.5 and pH 1.0, sialidase-Alcian Blue, pH 2.5, HID-AB pH 2.5) and lectin-histochemical (WGA, SWGA, PNA, sialidase-PNA, SBA, sialidase-SBA, DBA, sialidase-DBA, ConA, BSI-B4, AAA, UEA-1, LTA) techniques. Oesophageal lining epithelium consisted of ciliated and goblet cells, with no pluricellular glands. Mannosylated sialosulfomucins were observed. Fundic mucosa of stomach presented surface cells producing sialomucins with terminal sialic acid linked to galactose. In gastric glands neck and oxynticopeptic cells were found. Neck cells had sialomucins with mannose, N-acetylglucosamine, galactose, N-acetylgalactosamine and fucose-α-(1,2)-linked residues. Cytoplasm of oxynticopeptic cells showed N-acetylgalactosamine and fucose residues. Secretion of surface cells in pyloric mucosa was similar to that of fundic ones, differing in having fucose. Goblet cells in the small intestine of N. natrix produced sulfo- and sialomucins, with sialic acid linked to galactose and N-acetylgalactosamine residues. Mucins also presented residues of mannose. Goblet cells in the large intestine presented sulfomucins only, with terminal N-acetylgalactosamine, galactose and N-acetylglucosamine. The glycosylation patterns found are probably related to protection against injuries, gastric juice and microorganisms, both pathogenic and decomposers, as well as to dietary adaptations.

On the relationship between sialomucin and sulfomucin expression and hydrogenotrophic microbes in the human colonic mucosa.

The colonic mucus layer is comprised primarily of acidomucins, which provide viscous properties and can be broadly classified into sialomucins or sulfomucins based on the presence of terminating sialic acid or sulfate groups. Differences in acidomucin chemotypes have been observed in diseases such as colorectal cancer and inflammatory bowel disease, and variation in sialo- and sulfomucin content may influence microbial colonization. For example, sulfate derived from sulfomucin degradation may promote the colonization of sulfate-reducing bacteria (SRB), which through sulfate respiration generate the genotoxic gas hydrogen sulfide. Here, paired biopsies from right colon, left colon, and rectum of 20 subjects undergoing routine screening colonoscopies were collected to enable parallel histochemical and microbiological studies. Goblet cell sialo- and sulfomucins in each biopsy were distinguished histochemically and quantified. Quantitative PCR and multivariate analyses were used to examine the abundance of hydrogenotrophic microbial groups and SRB genera relative to acidomucin profiles. Regional variation was observed in sialomucins and sulfomucins with the greatest abundance of each found in the rectum. Mucin composition did not appear to influence the abundance of SRB or other hydrogenotrophic microbiota but correlated with the composition of different SRB genera. A higher sulfomucin proportion correlated with higher quantities of Desulfobacter, Desulfobulbus and Desulfotomaculum, relative to the predominant Desulfovibrio genus. Thus, acidomucin composition may influence bacterial sulfate respiration in the human colon, which may in turn impact mucosal homeostasis. These results stress the need to consider mucus characteristics in the context of studies of the microbiome that target intestinal diseases.

Tissue quantification of neutral and acid mucins in the mucosa of the colon with and without fecal stream in rats.

PURPOSE: To quantify the intensity of the expression of neutral and acids mucins in mucosa of the colon with and without fecal stream and to correlate this with the duration of fecal transit diversion. METHODS: Thirty male Wistar rats were subjected to fecal transit deviation in the left colon by a proximal colostomy and a distal mucous fistula. The animals were divided into three experimental groups, according to whether sacrificing would be performed six, 12 or 18 weeks after surgery. The expression of neutral and acid mucins was evaluated using the histochemical techniques of Periodic Acid Schiff and Alcian Blue, respectively. The tissue mucins expression was quantified by computer-assisted image analysis software (NIS-Elements) in the segments with and without fecal stream. Student's paired t test was used to compare the quantities of mucins in colon with or without fecal stream and variance between the experimental groups by ANOVA and Newman-Keuls post-test, establishing level of signification of 5% (p<0.05). RESULTS: There were significant decreased quantities of acid and neutral mucins in the colon without transit, compared with the colon with fecal stream, independent of the duration of exclusion. There was increased expression of neutral mucins in the colon with fecal stream after 12 and 18 weeks of exclusion. There was no increase in the expression of acid mucins in the colon with transit as the duration of fecal transit exclusion progressed. There was increased production of acid mucins in the animals submitted to diversion of the fecal stream for 18 weeks, compared with those subjected to diversion for 6 and 12 weeks. In the colon without fecal stream, there was increased expression of neutral mucins after 12 and 18 weeks of exclusion. CONCLUSIONS: Deviation of the fecal stream decreased the expression of acid and neutral mucins in the segments without fecal transit, compared with segments with transit. Regardless of the reduced expression of acid and neutral mucins in the segments without fecal stream, their tissue expression increased with increasing duration of intestinal deviation.

Tissue quantification of neutral and acid mucins in the mucosa of the colon with and without fecal stream in rats / Quantificação tecidual de mucinas neutras e ácidas na mucosa do cólon com e sem trânsito intestinal em ratos

PURPOSE: To quantify the intensity of the expression of neutral and acids mucins in mucosa of the colon with and without fecal stream and to correlate this with the duration of fecal transit diversion. METHODS: Thirty male Wistar rats were subjected to fecal transit deviation in the left colon by a proximal colostomy and a distal mucous fistula. The animals were divided into three experimental groups, according to whether sacrificing would be performed six, 12 or 18 weeks after surgery. The expression of neutral and acid mucins was evaluated using the histochemical techniques of Periodic Acid Schiff and Alcian Blue, respectively. The tissue mucins expression was quantified by computer-assisted image analysis software (NIS-Elements) in the segments with and without fecal stream. Student's paired t test was used to compare the quantities of mucins in colon with or without fecal stream and variance between the experimental groups by ANOVA and Newman-Keuls post-test, establishing level of signification of 5 percent (p<0.05). RESULTS: There were significant decreased quantities of acid and neutral mucins in the colon without transit, compared with the colon with fecal stream, independent of the duration of exclusion. There was increased expression of neutral mucins in the colon with fecal stream after 12 and 18 weeks of exclusion. There was no increase in the expression of acid mucins in the colon with transit as the duration of fecal transit exclusion progressed. There was increased production of acid mucins in the animals submitted to diversion of the fecal stream for 18 weeks, compared with those subjected to diversion for 6 and 12 weeks. In the colon without fecal stream, there was increased expression of neutral mucins after 12 and 18 weeks of exclusion. CONCLUSIONS: Deviation of the fecal stream decreased the expression of acid and neutral mucins in the segments without fecal transit, compared with segments with transit. Regardless of ...

OBJETIVO: Quantificar a intensidade de expressão de mucinas neutras e ácidas na mucosa cólica provida e desprovida de trânsito intestinal relacionando-a ao tempo de exclusão fecal. MÉTODOS: Trinta ratos Wistar machos foram submetidos à derivação do trânsito no cólon esquerdo por colostomia proximal e fístula mucosa distal. Os animais foram divididos em três grupos experimentais segundo o sacrifício ter sido realizado seis, 12 e 18 semanas após a cirurgia. A avaliação da expressão de mucinas neutras e ácidas na mucosa cólica foi realizada com as técnicas histoquímicas do Periódico Ácido de Schiff e Azul de Alcian, respectivamente. A quantificação da expressão tecidual das mucinas foi com auxílio de programa de análise de imagem assistida por computador (NIS-Elements) nos segmentos providos e desprovidos de trânsito fecal. Utilizou-se o teste t de Student pareado na comparação da expressão de mucinas nos segmentos com e sem trânsito e a variação na expressão entre os grupos experimentais pelo teste ANOVA e pós-teste de Newmann-Keuls, estabelecendo-se nível de significância de 5 por cento (p<0,05). RESULTADOS: Houve redução na quantidade de mucinas neutras e ácidas no cólon desprovido de trânsito quando comparado ao cólon provido de trânsito, independente do tempo de exclusão. Ocorreu aumento na expressão de mucinas neutras no cólon provido de trânsito intestinal após 12 e 18 semanas de exclusão. Não houve aumento na expressão de mucinas ácidas no cólon provido de trânsito com o progredir do tempo exclusão de trânsito fecal. Ocorreu aumento na produção de mucinas ácidas nos segmentos com exclusão de trânsito por 18 semanas quando comparados aos animais submetidos à exclusão por seis e 12 semanas. No cólon desprovido de trânsito ocorreu aumento na expressão de mucinas neutras após 12 e 18 semanas de exclusão. CONCLUSÕES: A derivação do trânsito fecal diminui a expressão de mucinas ácidas e neutras nos segmentos desprovidos de trânsito fecal ...