# Molecular mechanisms for sorting lysosomal proteins

> **NIH NIH R01** · VAN ANDEL RESEARCH INSTITUTE · 2022 · $475,000

## Abstract

Project Summary
The objective of this proposal is to gain mechanistic and pathological understanding of human GlcNAc-1-
phosphotransferase (PTase), an enzyme which modifies lysosomal hydrolases. Lysosomes contain several
dozen acid hydrolases that break down unwanted proteins and lipids. Lysosomal enzymes acquire a mannose-
6-P moiety in the Golgi apparatus by the action of PTase, which uses UDP-GlcNAc as a donor substrate. The
mannose-6-P functions like a zip code that guides the delivery of these enzymes to the lysosomes. PTase is
encoded by the GNPTAB and GNPTG genes and assembles a 340-kDa heterohexamer of α2β2γ2. PTase
selectively modifies lysosomal (but not non-lysosomal) glycoproteins transiting the ER–Golgi system. It has
been unclear how PTase catalyzes the GlcNAc-P transfer reaction and how it distinguishes lysosomal from
non-lysosomal glycoproteins. Mutations in the genes encoding PTase cause mucolipidosis II and III and lead to
abnormal body structure, mental retardation, and several neurological diseases. In preliminary studies, we
have determined the atomic structure of the catalytic core of the dimeric α2β2 subcomplex in the apo form. We
have identified an inhibitory hockey stick motif that moves in and out of the catalytic pocket in the absence of
substrates. We have also derived a nm-resolution cryo-EM 3D map of the holoenzyme. Building on these
strong preliminary studies, we propose to determine the structures of the catalytic core bound to the donor
substrate UDP-GlcNAc and the acceptor substrate mimic α-methylmannoside, and to perform structure-guided
mutagenesis and in vitro activity assays. These structure-function studies of the catalytic core will enable us to
formulate the unique phospho-glycosyl transfer reaction mechanism. We also propose to carry out systematic
mutational and functional assays to examine the many reported disease-causing mutations. Finally, we
propose to study the structures of a large lysosomal hydrolase-binding peripheral region and the γ-subunit of
the PTase holoenzyme and to explore how they interacts with selected substrate lysosomal hydrolases. The
proposed research will address the molecular mechanism of a key signaling pathay in glycobiology and will
provide molecular insights into how mutations in the PTase cause the associated human diseases.

## Key facts

- **NIH application ID:** 10521596
- **Project number:** 1R01NS127292-01A1
- **Recipient organization:** VAN ANDEL RESEARCH INSTITUTE
- **Principal Investigator:** Huilin Li
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $475,000
- **Award type:** 1
- **Project period:** 2022-07-10 → 2027-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10521596

## Citation

> US National Institutes of Health, RePORTER application 10521596, Molecular mechanisms for sorting lysosomal proteins (1R01NS127292-01A1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10521596. Licensed CC0.

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