# Biology of α-linked glycosylceramides in the immune system

> **NIH NIH R01** · SCRIPPS RESEARCH INSTITUTE, THE · 2020 · $723,259

## Abstract

Project Summary
As cellular adjuvants of immunity, NKT cells prime and control the maturation of dendritic cells during all
immune responses. Therefore, the understanding of the biology of these regulatory cells should lead to their
utilization in anti-microbial and cancer vaccines, immunotherapy of chronic infectious diseases, and the
prevention of infection in at-risk patients. We have worked for the past two decades on the identification of the
natural endogenous ligands of NKT cells thinking that they could be manipulated in vivo. By creating new tools
and approaches, we have recently succeeded in demonstrating that α-glycosylceramides were the ligands of
NKT cells. Since publication of our work, and the first submission of this proposal, we have made great
progress in understanding this new class of glycolipids for which each metabolic step is to be described. In the
thymus, αGalcer is produced by DCs in the medullary region, whereas αGlucer is made by cortical thymocytes.
To understand the respective roles of each ligand in thymic selection and peripheral activation, we are using
CRISPR/Cas9 technology to deconstruct the pathways of synthesis and degradation. Galactose mutarotase
(Galm) for αGalcer and glucose ceramide synthase (UGCG) for αGlucer are necessary for synthesis; acid
ceramidase, acid α galactosidase, acid α glucosidase, and catheptsin L are essential for degradation. A
powerful new mass spectrometry technique should allow us to measure directly α anomers from cells and
tissues. As the work moves from cell lines to in vivo studies, our labor is divided in three specific aims: Aim 1:
Biosynthesis of endogenous α-galactosylceramides. Following CRISPR/Cas9 gene inactivation, a robust set of
phenotypic and functional assays has been implemented to evaluate the role of each molecule in the pathway.
The consequences of the elimination of αGalcer for thymic selection, shaping of the NKT cell repertoire, and
peripheral functions will be evaluated in Galm knockout mice. Aim 2: Biosynthesis of endogenous α-
glucosylceramides. The screen of enzymes and transporters involved in the synthesis of glucosylceramides
using CRISPR/Cas9 technology has revealed the role of UGCG. Its role as a synthase or the necessity to use
an anomerase in a two step synthesis will be explored. The functional consequences of eliminating αGlucer
will be evaluated. Aim 3: Catabolism of α-linked glycosylceramides. Both αGlucer and αGalcer catabolisms are
controlled by acid ceramidase, ASAH1, which cleaves off the fatty acid to produce α-lysoglycosylceramides, a
family of potent NKT cell activators. We will evaluate the biological role of these lysolipids by studying their
inactivation when α-linked glucose and galactose are removed by acid α glycosidases in the lysosome.
Cathepsin L functions in processing and activating these various hydrolases will also be examined in cells and
animals. The manipulation of degradation of αGlucer and αGalcer by using specific enzyme...

## Key facts

- **NIH application ID:** 9839474
- **Project number:** 5R01AI123130-04
- **Recipient organization:** SCRIPPS RESEARCH INSTITUTE, THE
- **Principal Investigator:** Luc Teyton
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $723,259
- **Award type:** 5
- **Project period:** 2017-01-02 → 2021-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9839474

## Citation

> US National Institutes of Health, RePORTER application 9839474, Biology of α-linked glycosylceramides in the immune system (5R01AI123130-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9839474. Licensed CC0.

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