Role of OligoSaccharyl Transferase enzymes in developmental signaling and congenital disorders of glycosylation

Project Information

OSTers

Grant agreement ID: 844147

DOI

10.3030/844147

Project closed

EC signature date 20 March 2019

Start date 1 June 2019

End date 31 May 2021

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 183 473,28

EU contribution

€ 183 473,28

Coordinated by

UNIVERSITA DEGLI STUDI DI MILANO
Italy

Periodic Reporting for period 1 - OSTers (Role of OligoSaccharyl Transferase enzymes in developmental signaling and congenital disorders of glycosylation)

Reporting period: 2019-06-01 to 2021-05-31

Summary of the context and overall objectives of the project

The action” Role of OligoSaccharyl Transferase enzymes in developmental signaling and congenital disorders of glycosylation” focuses on genetic defects affecting the assembly and addition of (N)-glycans to proteins. N-glycosylation occurs in a dedicated compartment of the cells, the endoplasmic reticulum, by the Oligosaccharyl transferase (OST) complex. This work investigated what is the molecular mechanism by which mutations in OST complex cause abnormal phenotypes in animal development to decipher some aspects of patients’ clinical features with Congenital disorders of glycosylation (CDGs). The fellow conducting this research paid particular interest in the basic biology of glycosylation-related genes and how they affect protein folding, protein trafficking, and ultimately cell signaling.

Recent advances in genome sequencing technologies are increasing the pace of discovery for new types of genetic diseases associated with glycosylation, opening questions on the biological relevance of the enzymes involved in the glycosylation machinery and how the disruption of such genes results in disease remains a constant challenge. Given the unknown nature of pathogenesis of the glycosylation-linked diseases, the research conducted in this MSCA action provides a link between the basic biology of glycosylation and clinical features of CGD patients for allowing the transfer of research knowledge to clinical practice. Importantly, understanding the role of glycosylation genes in cell functions offers useful insights not only for future therapies of CDG patients but also for patient stratification and monitoring, such as in cancer, nutrition, and immunology fields.

Objectives of this Marie Skłodowska Curie Action (MSCA) have been to (a) characterize and compare the roles of OST complex during development in disease models; (b) understand the molecular basis for the regulation of developmental signaling pathway by N-glycosylation; (c) characterize the dysregulation of OST complex in mammalian cell models and OST-linked diseases. As a personal objective, the MSCA Individual Fellowship (MSCA-IF) has developed my potential for an advanced career, both in academic and entrepreneurial sectors connected to congenital rare diseases. Therefore MSCA-IF helped me to establish new collaborations with prominent scientists, with special attention to the Italian network, along with patient associations in the spirit of a synergic knowledge-building environment.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The MSCA action included 4 work packages (WPs). WP1 determined the role of Stt3A and OSTγ (two components of OST complex) during Drosophila development, as a model of OST-linked diseases. The results indicate that of Stt3A and OSTγ (the fly homolog of human STT3A and TUSC3, respectively) are essential genes during Drosophila development and both genes are required early to ensure animal survival. In particular, adult lethality is likely ascribed to their loss in neurons and mesodermal cells with a special dependency on BMP signaling. WP2 sought to determine the molecular basis for the regulation of fly BMP signaling by Stt3A and OSTγ. The main results indicate that OSTγ is required in fly midgut for ensuring BMP signaling. Its role is to mask the N-glycosylation sites in the mature domain of Dpp (the fly homolog of human BMP4) and prevents its glycosylation. WP3 validated in a mammalian cell model and OST-linked diseases the results obtained in the OST-disease fly model. The main results provide pieces of evidence for the molecular mechanism of TUSC3 function in BMP signaling. Specifically, upon loss of TUSC3 misfolded BMP4 molecules accumulate in the ER upstream of the folding quality control machinery. Based on the observations, I envisage that TUSC3 could be a part of regulatory machinery that shunts polypeptides that cannot properly be folded, perhaps because their abundance saturates the folding machinery, toward ERAD degradation. In this model, the role of TUSC3 could be to mask the glycosylation site on the polypeptide by forming a cysteine bond, as previously reported. This event could then induce Malectin to bind the un- or mis-glycosylated substrate to ultimately send it to the retrotraslocon. In WP4, the Fellow implemented his research skills in particular sharing knowledge with the host organization in particular with the supervisor’s group, Thomas Vaccari laboratories. Moreover, the Fellow learned how to manage reagent offers and the interface with vendors and chemical and biological companies with the help of the head of the Project Offices and department offices of the University of Milan. By attending weekly group meetings with the Vaccari group and monthly presenting his data, the Fellow implemented his communication skills in a knowledge-building environment. This all allowed the Fellow to join 5 conferences (3 as invited speakers), 8 workshops, advice and mentor two full-time Master Degree students, and attend several technical trainings.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The results of this project lead to developing a framework for understanding the mechanisms by which N-glycosylation mediates developmental signaling pathways. This MSC action allowed me to use cutting-edge technologies in animal and cell models for studying the molecular mechanism by which mutations in glycosylation-related genes cause some aspects of patients’ clinical features with Congenital disorders of glycosylation (CDGs). This research has pushed the frontiers forward for identifying a novel role of N-glycosylation machinery. Notably, the data suggest that components of the glycosylation machinery could be associated with a novel degradation pathway mostly devoted to too high and fast protein synthesis at ER-associated ribosomes. The understanding of N-glycosylation initial steps can be crucial not only for deciphering the CDG features but also relevant in other diseases that are reported to depend on protein folding.
Along with the scientific impact of the project, my work has established local and international networks with scientists involved in the glycosylation field. I advertised my results in 4 major communication events, organized by Italian and European public awareness conferences following the motto “Rare is not an excuse” that comes from the world campaign for curing patients with undiagnostic or rare diseases. My engagement for knowledge dissemination to the public has been in the line with the Responsible Research and Innovation (RRI) guideline from the European Commission. I hope that the publication of part of this work in a relevant journal, the workshops, and community up-skilling events: (1) stimulate pharmaceutical companies to make available potential drugs for curing CDG patients; (2) increase the approach to other medical emergencies, such cancer, which can be view as a collection of rare diseases; (3) engage young scientists to embark careers in research of CDG for progressing basic biology knowledge.

Vaccari Lab Summer party 2021

June 2019 Vaccari Lab retreat in Budapest

LeBiotech2020_invited speaker