In a groundbreaking study published in iScience on October 28th, 2025, researchers have unveiled novel insights into the selective modification of glycoprotein substrates by the enzyme N-acetylglucosaminyltransferase-V (GnT-V) within mouse kidney tissue. Glycans, complex carbohydrates decorating the surfaces of cells, play pivotal roles in intercellular communication, structural integrity, and protection against environmental insults. The nuanced attachment of these carbohydrates to proteins—a process known as glycosylation—varies significantly between proteins, influencing cellular behavior and disease pathology. The work spearheaded by Yasuhiko Kizuka from Gifu University delves into the enigmatic selectivity exhibited by GnT-V, an enzyme frequently upregulated in cancer and linked to a spectrum of diseases including Alzheimer’s, emphysema, diabetes, and oncogenesis.
Glycosylation, a ubiquitous post-translational modification, occurs principally through two varieties: N-glycosylation and O-glycosylation. This study hones in on N-glycosylation, wherein glycans are attached to the nitrogen atom of asparagine residues in proteins. The researchers undertook an in-depth analysis to decipher how GnT-V, known for synthesizing branched N-glycan structures associated with tumor progression, discerns its glycoprotein substrates amidst the cellular milieu. Despite the ubiquity of GnT-V substrates, the enzyme’s preferential modification patterns remained poorly understood prior to this investigation.
Employing mouse kidney epithelial cells as a polarized cellular model, the study demonstrates that GnT-V’s substrate selectivity is governed not merely by the linear amino acid sequences of target proteins but is profoundly influenced by the three-dimensional conformation of these proteins and their intracellular trafficking patterns. Polarized cells, characterized by distinct apical and basal membrane domains, present unique spatial challenges for enzymatic modification. The apical and basal surfaces perform divergent physiological roles, and this cellular compartmentalization appears to play a decisive role in substrate recognition by GnT-V.
The researchers identified two metalloproteases—enzymes responsible for proteolytic cleavage through metal ion cofactors—as primary glycoprotein substrates predominantly localized on the apical surface of kidney epithelial cells. The colocalization of these substrates with GnT-V within the apical compartment argues that intracellular trafficking routes selectively direct these proteins toward Golgi apparatus regions where GnT-V activity prevails. This spatial confinement suggests that the enzyme’s substrate specificity arises from a confluence of protein architecture and predetermined intracellular processing routes.
Crucially, the data support a model wherein GnT-V’s catalytic activity is spatially regulated within polarized cells, targeting proteins as they transit the secretory pathway to the apical surface. Such compartmentalized enzymatic action not only enhances substrate specificity but could also modulate the functional glycan landscapes that influence cell signaling, adhesion, and immune recognition. This mechanism adds a new dimension to our understanding of how glycan heterogeneity arises despite the broad substrate availability.
However, the study also underscores inherent limitations, particularly the reliance on specific protein markers to isolate glycoprotein substrates. This approach, while precise, raises the possibility that other relevant substrates could remain unidentified. Additionally, because the experimental system involves polarized kidney cells, extrapolation to non-polarized tissues or organs with differing cellular architectures warrants cautious interpretation. Whether GnT-V’s substrate selectivity is universally influenced by cell polarity remains an open question.
Notwithstanding these limitations, the implications of this research extend far beyond kidney physiology. The aberrant upregulation of GnT-V is a hallmark in diverse malignancies, where altered glycosylation patterns foster tumor progression, metastasis, and immune evasion. A deeper mechanistic understanding of GnT-V’s substrate discrimination may trigger a paradigm shift in the design of glycan-targeted therapeutics and diagnostics. Targeting the enzyme’s selective activity could enable precise remodeling of glycan structures to restore normal cellular function or impede pathological processes.
Yasuhiko Kizuka emphasizes the therapeutic promise that stems from decoding the rules governing glycosylation enzyme specificity. “This could lead to the precise prediction of glycan structures of each glycoprotein in cells, contributing to eventual remodeling of glycans for therapeutic purposes,” he stated. Such advancements may pave the way for novel interventions in cancer, neurodegenerative diseases, and other glycan-related disorders by tailoring enzyme activity or glycan presentation.
The study itself represents a collaborative success among multiple Japanese institutions, including the United Graduate School of Agricultural Science at Gifu University, Osaka University, Hiroshima University, Kumamoto University, Fujita Health University School of Medicine, and the Institute for Glyco-core Research (iGCORE). Funding support came from prestigious agencies such as the Japan Science and Technology Agency, Japan Society for the Promotion of Science, Japan Agency for Medical Research and Development, as well as initiatives like the Human Glycome Atlas project.
In technical terms, the comprehensive experimental approach combined advanced glycoproteomics, confocal imaging of polarized cells, and structural protein analyses to tease apart the determinants of substrate recognition. The integration of subcellular localization data with enzymatic activity profiles highlights a sophisticated orchestration of glycan biosynthesis within cellular microenvironments, challenging the previously held assumption of random or solely sequence-based glycosyltransferase activity.
Future investigations are expected to broaden the understanding of GnT-V beyond the confines of kidney tissues, probing its behavior in different cellular contexts and pathological conditions. Moreover, dissecting the molecular signals that direct protein trafficking to GnT-V-rich Golgi subdomains may reveal novel regulatory nodes suitable for pharmacological intervention. The pursuit of these questions stands to accelerate progress in glycobiology and its translational applications.
This landmark study offers a compelling narrative that links protein structure, intracellular organization, and enzymatic selectivity into a coherent framework, enriching our comprehension of glycan biosynthesis. By illuminating the selective modification strategies of GnT-V, researchers have opened new avenues for exploiting glycosylation in disease diagnostics and therapy, underscoring the critical role of carbohydrate biology in health and disease.
Subject of Research: Cells
Article Title: Selective modification of glycoprotein substrates by GnT-V in mouse kidney
News Publication Date: 28-Oct-2025
Web References: DOI: 10.1016/j.isci.2025.113894
Image Credits: Yasuhiko Kizuka, Institute for Glyco-core Research (iGCORE), Gifu University
Keywords: Life sciences, Biochemistry, Glycobiology, Glycomics, Cell biology, Nephropathies
Tags: enzyme selectivity in glycosylationglycoprotein modification mechanismsglycosylation and neurodegenerative diseasesglycosylation in disease pathologyimplications for cancer researchintercellular communication and glycansmouse kidney tissue researchN-acetylglucosaminyltransferase-V functionN-glycosylation significanceselective glycosylation enzymesstructural integrity of glycoproteinstumor progression and glycosylation
