Key Takeaways & Executive Findings
- •• CD98hc is identified as a novel binding partner for galectin-8 (Gal-8), expanding the known repertoire of Gal-8 receptors. • The interaction between CD98hc and Gal-8 is N-glycosylation-dependent, as demonstrated by inhibition with tunicamycin and pull-down assays. • Both N- and C-terminal carbohydrate recognition domains (CRDs) of Gal-8 bind to CD98hc, with distinct affinities (Gal-8N: 0.22 μM; Gal-8C: 10.68 μM). • The binding is specifically inhibited by lactose, indicating β-galactoside specificity, and may have implications for Gal-8-mediated cellular functions.
Abstract
Glycan-mediated recognition plays a critical role in facilitating cell-cell and cell-matrix interactions. Galectin-8 (Gal-8), classified as a ‘tandem-repeat’ type of galectin, binds to cell surface glycans to modulate various cellular functions, including cell adhesion, migration, apoptosis, pathogen recognition, autophagy, and immunomodulation. Despite the known function of Gal-8 in binding to various glycosylated proteins, only a few interactions have been reported to date. In this study, mass spectrometry is used to identify CD98hc as a novel binding partner for Gal-8. Both the N-terminal and C-terminal carbohydrate recognition domains (CRDs) of Gal-8 (Gal-8N and Gal-8C) bind to CD98hc, an interaction that is specifically inhibited by lactose but not sucrose, as confirmed by pull-down assays. The binding affinity between CD98hc and Gal-8 measured by microscale thermophoresis (MST) is 1.51 ± 0.17 μM. In addition, Gal-8N and Gal-8C have the binding affinities of 0.22 ± 0.03 μM and 10.68 ± 1.69 μM, respectively. Gal-8N and Gal-8C are both involved in the recognition and binding process of CD98hc. Furthermore, both full-length Gal-8 and its individual CRDs bind specifically to N-glycosylated glycans on CD98hc, as demonstrated by the use of tunicamycin to inhibit N-glycosylation in cells. In addition, Gal-8 and its individual CRDs can pull down glycosylated CD98hc-ED but not free CD98hc-ED in vitro, indicating that the binding of Gal-8 to glycosylated CD98hc-ED is N-glycosylation-dependent. Overall, our findings establish CD98hc as a novel binding partner for Gal-8 and provide insights for further exploration of the diverse biological functions of Gal-8.
1. Introduction
Protein glycosylation, which is crucial to cell and tissue physiology, is highly complex and regulates numerous pathological disorders, and interactions between various cells and their environments are partially dependent on glycosylation [1]. Galectin-8 (Gal-8) belongs to a family of lectins that generally bind to β-galactoside-based glycoconjugates [2–4]. Galectins are generally divided into three groups: (i) prototype galectins (Gal-1, -2, -5, -7, -10, -11, -13, -14, -15 and -16) with a single carbohydrate recognition domain (CRD), (ii) tandem-repeat galectins (Gal-4, -6, -8, -9, and -12) with two different CRDs joined by a linker peptide, and (iii) chimera-type Gal-3 with a single CRD linked to a long, aperiodic and proline-rich N-terminal tail [4–6]. As a tandem-repeat galectin, Gal-8 is composed of N- and C-CRDs connected by a peptide linker of variable length [7–9]. The N- and C-CRDs of Gal-8 are approximately 35% homologous, whereas their glycan specificities differ significantly. This bifunctionality makes it possible for Gal-8 to cross-link a wide variety of glycoconjugates, contributing to the functional diversity of Gal-8 [10,11].
Compared with other tandem-repeat galectins, Gal-8 is more widely distributed in mammalian tissues, such as the liver, heart, muscles, kidney, and brain [12]. Gal-8 lacks a signal peptide and is thus secreted extracellularly via a nonclassical pathway. This process involves Gal-8 accumulating on the cytoplasmic side of the plasma membrane and being released in vesicles and exosomes or translocating directly through the plasma membrane [13]. In the extracellular space, Gal-8 binds to cell surface glycoproteins that trigger a downstream signaling cascade with various receptors, such as FAK, ERK, and JNK [12]. This, in turn, modulates various cellular functions, from cell adhesion, migration, and apoptosis to pathogen recognition, autophagy, and immunomodulation [12,14,15]. These findings underscore the biological importance of Gal-8.
As a major class of cell surface adhesion receptors, integrins (and other adhesion receptors) bind to Gal-8 to promote cell adhesion [16–19]. Several of these receptors are known. For example, CD44vRA is a variant of CD44 that has high affinity for Gal-8. In rheumatoid arthritis (RA), the formation of this complex influences the extent of the autoimmune inflammatory response [20]. CD166, also known as activated leukocyte cell adhesion molecule (ALCAM), is overexpressed in various tumors, and its expression level and cell surface abundance are correlated with poor prognosis [21]. CD166 is a Gal-8-binding partner involved in the regulation of cell adhesion and migration of tumor cells, as well as endothelial cell-based angiogenesis, processes that are critical to tumor progression [22,23]. Podoplanin (PDPN) is a heavily O-glycosylated, small mucin-type transmembrane glycoprotein with a wide variety of functions.
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Yunlong Si, Jiahui Zhu, Hend Sayed, Kevin H. Mayo, Yifa Zhou, Guihua Tai, Jiyong Su (2026). CD98hc, a novel of galectin-8 receptor, binds to galectin-8 in an N-glycosylation-dependent manner. Acta Biochimica et Biophysica Sinica. https://doi.org/10.3724/abbs.2024182
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Frequently Asked Questions
What is the main finding of this study?
The study identifies CD98hc as a novel binding partner for galectin-8 (Gal-8), and demonstrates that the interaction is dependent on N-glycosylation of CD98hc.
How was the binding between CD98hc and Gal-8 characterized?
The binding was characterized using pull-down assays, microscale thermophoresis (MST), and inhibition studies with lactose and tunicamycin. The affinity was measured at 1.51 ± 0.17 μM for full-length Gal-8, with individual CRDs showing different affinities.
What is the significance of the N-glycosylation dependence?
The N-glycosylation dependence indicates that the interaction is specific to glycosylated forms of CD98hc, which may have implications for understanding Gal-8-mediated cellular functions in health and disease.
Which domains of Gal-8 are involved in binding to CD98hc?
Both the N-terminal (Gal-8N) and C-terminal (Gal-8C) carbohydrate recognition domains are involved, with Gal-8N showing higher affinity (0.22 μM) compared to Gal-8C (10.68 μM).
What are the potential applications of this research?
This research may contribute to understanding the molecular mechanisms of Gal-8 in cell adhesion, migration, and immune modulation, and could inform therapeutic strategies targeting Gal-8-CD98hc interactions.
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