Charlotte Jeanneau

Polypeptide GalNAc-transferase T3 and Familial Tumoral Calcinosis SECRETION OF FIBROBLAST GROWTH FACTOR 23 REQUIRES O-GLYCOSYLATION

Mutations in the gene encoding the glycosyltransferase polypeptide GalNAc-T3, which is involved in initiation of O-glycosylation, were recently identified as a cause of the rare autosomal recessive metabolic disorder familial tumoral calcinosis (OMIM 211900). Familial tumoral calcinosis is associated with hyperphosphatemia and massive ectopic calcifications. Here, we demonstrate that the secretion of the phosphaturic factor fibroblast growth factor 23 (FGF23) requires O-glycosylation, and that GalNAc-T3 selectively directs O-glycosylation in a subtilisin-like proprotein convertase recognition sequence motif, which blocks processing of FGF23. The study suggests a novel posttranslational regulatory model of FGF23 involving competing O-glycosylation and protease processing to produce intact FGF23.

Current trends in the structure–activity relationships of sialyltransferases

Sialyltransferases (STs) represent an important group of enzymes that transfer N-acetylneuraminic acid (Neu5Ac) from cytidine monophosphate-Neu5Ac to various acceptor substrates. In higher animals, sialylated oligosaccharide structures play crucial roles in many biological processes but also in diseases, notably in microbial infection and cancer. Cell surface sialic acids have also been found in a few microorganisms, mainly pathogenic bacteria, and their presence is often associated with virulence. STs are distributed into five different families in the CAZy database (http://www.cazy.org/). On the basis of crystallographic data available for three ST families and fold recognition analysis for the two other families, STs can be grouped into two structural superfamilies that represent variations of the canonical glycosyltransferase (GT-A and GT-B) folds. These two superfamilies differ in the nature of their active site residues, notably the catalytic base (a histidine or an aspartate residue). The observed structural and functional differences strongly suggest that these two structural superfamilies have evolved independently.

Changes in the profile of simple mucin-type O-glycans and polypeptide GalNAc-transferases in human testis and testicular neoplasms are associated with germ cell maturation and tumour differentiation

Testicular germ cell tumours (TGCT) exhibit remarkable ability to differentiate into virtually all somatic tissue types. In this study, we investigated changes in mucin-type O-glycosylation, which have been associated with somatic cell differentiation and cancer. Expression profile of simple mucin-type O-glycans (Tn, sialyl-Tn, T), histo-blood group H and A variants and six polypeptide GalNAc-transferases (T1–4, T6, T11) that control the site and density of O-glycosylation were analysed by immunohistochemistry during human testis development and in TGCT. Normal testis showed a restricted pattern; gonocytes expressed abundant sialyl-Tn and sialyl-T, and adult spermatogonia were devoid of any glycans, whereas spermatocytes and spermatids expressed exclusively glycans Tn and T and the GalNAc-T3 isoform. A subset of mature ejaculated spermatozoa expressed an additional glycan sialyl-T. The pattern found in testicular neoplasms recapitulated the developmental order: Pre-invasive carcinoma in situ (CIS) cells and seminoma expressed fetal type sialylated glycans in keeping with their gonocyte-like phenotype. Neither simple mucin-type O-glycans nor GalNAc-transferase isoforms were found in undifferentiated nonseminoma, i.e. embryonal carcinoma, whereas teratomas expressed them all to some extent but in a disorganized manner. We concluded that simple mucin-type O-glycans and their transferases are developmentally regulated in the human testis, with profound changes associated with neoplasia. The restricted O-glycosylation pattern in haploid germ cells suggests a role in their maturation or egg recognition/fertilization warranting further studies in male infertility, whereas the findings in TGCT provide new diagnostic tools and support our hypothesis that testicular cancer is a developmental disease of germ cell differentiation.

Complement Activation by Pulp Capping Materials Plays a Significant Role in Both Inflammatory and Pulp Stem Cells' Recruitment

Introduction: The role of complement, especially through the C5a fragment, is well‐known for the initiation of inflammation. Its involvement in regeneration has been shown more recently by the recruitment of mesenchymal stem cells. C5a can be produced locally by the pulp fibroblasts in response to injury or infection. This work aims to investigate the effect of different pulp capping biomaterials on complement activation and its possible influence on inflammatory and pulp stem cell recruitment. Methods: Conditioned media were prepared from 3 pulp capping biomaterials: Biodentine (Septodont, Saint‐Maur‐des‐Fosses, France), TheraCal (BISCO, Lançon De Provence, France), and Xeno III (Dentsply Sirona, Versaille, France). Injured pulp fibroblasts were cultured with these conditioned media to analyze C5a secretion using an enzyme‐linked immunosorbent assay. Dental pulp stem cells (DPSCs) were isolated from human third molar explants by magnetic cell sorting with STRO‐1 antibodies. The expression of C5a receptor on DPSCs and inflammatory (THP‐1) cells was investigated by immunofluorescence. The migration of both DPSCs and THP‐1 cells was studied in Boyden chambers. Results: Pulp fibroblast production of C5a significantly increased when the cells were incubated with TheraCal‐ and Xeno III–conditioned media. The recruitment of cells involved in inflammation (THP‐1 cells) was significantly reduced by Biodentine‐ and TheraCal‐conditioned media, whereas the migration of DPSCs was reduced with TheraCal‐ and Xeno III–conditioned media but not with that of Biodentine. The involvement of C5a in cell recruitment is demonstrated with a C5a receptor–specific antagonist (W54011). Conclusions: After pulp injury, the pulp capping material affects complement activation and the balance between inflammation and regeneration through a differential recruitment of DPSCs or inflammatory cells. HighlightsPulp capping materials affect C5a secretion by injured pulp fibroblasts.Biodentine reduces inflammatory cell migration without affecting that of DPSCs.TheraCal reduces both inflammatory cells and dental pulp stem cell migration.The biomaterial influences the balance between inflammation and regeneration.

Dental Pulp Stem Cell Recruitment Signals within Injured Dental Pulp Tissue

The recruitment of dental pulp stem cells (DPSC) is a prerequisite for the regeneration of dentin damaged by severe caries and/or mechanical injury. Understanding the complex process of DPSC recruitment will benefit future in situ tissue engineering applications based on the stimulation of endogenous DPSC for dentin pulp regeneration. The current known mobilization signals and subsequent migration of DPSC towards the lesion site, which is influenced by the pulp inflammatory state and the application of pulp capping materials, are reviewed. The research outcome of migration studies may be affected by the applied methodology, which should thus be chosen with care. Both the advantages and disadvantages of commonly used assays for investigating DPSC migration are discussed. This review highlights the fact that DPSC recruitment is dependent not only on the soluble chemotactic signals, but also on their interaction with neighboring cells and the extracellular matrix, which can be modified under pathological conditions. These are discussed to explain how these modifications lead to the stimulation of DPSC recruitment.

Expression of the O-Glycosylation Enzyme GalNAc-T3 in the Equatorial Segment Correlates with the Quality of Spermatozoa

We question whether the expression of GalNAc-T3, the only known O-GalNAc-transferase present in germ cells, is correlated with qualitative and functional parameters of spermatozoa. We investigated the expression of GalNAc-T3 in ejaculated spermatozoa with immunocytochemistry in swim-up purified and acrosome-reacted spermatozoa from quality-control semen donors and in semen samples from 206 randomly selected men representing a broad spectrum of semen quality. Using donor ejaculates and immunofluorescence detection we found that expression of GalNAc-T3 and the presence of the immature O-glycans Tn and T localized to the equatorial segment of spermatozoa. The proportion of GalNAc-T3-positive spermatozoa in the ejaculate increased after swim-up and appeared unaffected by induction of acrosomal exocytosis. The fraction of spermatozoa with equatorial expression of GalNAc-T3 correlated with classical semen parameters (concentration p = 9 × 10−6, morphology p = 7 × 10−8, and motility p = 1.8 × 10−5) and was significantly lower in men with oligoteratoasthenozoospermia (p = 0.0048). In conclusion, GalNAc-T3 was highly expressed by motile spermatozoa and the expression correlated positively with the classical semen parameters. Therefore, GalNAc-T3 expression seems related to the quality of the spermatozoa, and we propose that reduced expression of GalNAc-T3 may lead to impaired O-glycosylation of proteins and thereby abnormal maturation and reduced functionality of the spermatozoa.

Pulp capping materials modulate the balance between inflammation and regeneration

The interrelations between inflammation and regeneration are of particular significance within the dental pulp tissue inextensible environment. Recent data have demonstrated the pulp capacity to respond to insults by initiating an inflammatory reaction and dentin pulp regeneration. Different study models have been developed in vitro and in vivo to investigate the initial steps of pulp inflammation and regeneration. These include endothelial cell interaction with inflammatory cells, stem cell interaction with pulp fibroblasts, migration chambers to study cell recruitment and entire human tooth culture model. Using these models, the pulp has been shown to possess an inherent anti-inflammatory potential and a high regeneration capacity in all teeth and at all ages. The same models were used to investigate the effects of tricalcium silicate-based pulp capping materials, which were found to modulate the pulp anti-inflammatory potential and regeneration capacity. Among these, resin-containing materials such as TheraCal® shift the pulp response towards the inflammatory reaction while altering the regeneration process. On the opposite, resin-free materials such as Biodentine™ have an anti-inflammatory potential and induce the pulp regeneration capacity. This knowledge contradicts the new tendency of developing resin-based calcium silicate hybrid materials for direct pulp capping. Additionally, it would allow investigating the modulatory effects of newly released pulp capping materials on the balance between tissue inflammation and regeneration. It would also set the basis for developing future capping materials targeting these processes.

Response to Letter to the Editor, “The Role of Membrane Attack Complex Formation against Gram-positive Bacteria”

It has long been accepted that Gram-positive bacteria resist the membrane attack complex (MAC) because of a protective, thick peptidoglycan layer (Joiner et al. 1983). However, this is contradicted by reports showing that MAC components are assembled on Gram-positive bacteria in specific localizations and that this assembly differs among bacterial strains (Berends et al. 2013). Also, MAC components’ deficiency increases patients’ susceptibility to infections not only with Gram-negative but also with some Gram-positive bacteria (Skattum et al. 2011). Moreover, many Gram-positive bacteria not only recruit the host inhibitors of MAC but also express proteins that inhibit MAC formation (Laursen et al. 2010). For example, Streptococcus pyogenes secretes streptococcal inhibitor of complement, which blocks the C5b-7 membrane insertion site and thereby prevents MAC formation (Akesson et al. 1996). This shows clearly that the peptidoglycan wall is not enough to protect all Gram-positive bacteria from MAC lysis.