The Essential Role of the PA14 Domain in B4GALNT3 and the Regulatory Impact of LacdiNAc on N-Glycan Biosynthesis

Glycosylation, the process of attaching sugar molecules to proteins or lipids is a key biological mechanism that impacts countless cellular processes from immune responses to cell communication and even the clearance of proteins from the bloodstream. However, despite its importance, we still do not fully understand how this complex process works and there are many unanswered questions about how specific glycan structures—complex sugar chains—are created and how they influence various biological systems. Among these structures, LacdiNAc (GalNAcβ1-4GlcNAc) is especially intriguing because of its distinct roles in regulating protein behavior. It has been linked to critical processes like stem cell maintenance, bone health, and the turnover of glycoproteins. LacdiNAc is synthesized by two enzymes, B4GALNT3 and B4GALNT4, which modify specific glycan structures on proteins. While the more common LacNAc (Galβ1-4GlcNAc) is often used as a base for terminal modifications like sialylation or fucosylation, LacdiNAc serves different purposes. For instance, it can control how quickly proteins are cleared from the bloodstream or help maintain the stemness of certain cells. Despite its significance, researchers have struggled to fully understand how LacdiNAc is synthesized and why it only appears on specific proteins. Another challenge is understanding how B4GALNT3 and B4GALNT4 work at a molecular level. Scientists are still trying to uncover how these enzymes recognize their substrates and what structural features allow them to function. Another puzzle involves how LacdiNAc affects other glycan modifications, such as the addition of sialic acid or fucose, and how it interacts with bisecting GlcNAc—a modification that seems to inhibit LacdiNAc formation, and vice versa.

To address these challenges, a new study published in the Journal of Biological Chemistry led by Professor Yasuhiko Kizuka and Yuko Tokoro from Gifu University together with Dr. Masamichi Nagae from Osaka University, Dr. Miyako Nakano from Hiroshima University and Dr. Anne Harduin-Lepers from University of Lille, shed light on these issues and focused on B4GALNT3. The research team began by using structural modeling with AlphaFold2 to analyze the enzyme B4GALNT3 and found that B4GALNT3 has a special PA14 domain, a region that does not directly catalyze reactions but is likely crucial for recognizing glycans and helping the enzyme function properly. To test this theory, the team created a mutant version of B4GALNT3 that lacked the PA14 domain and then expressed this mutant in HEK293 cells. What they observed was striking—the mutant enzyme showed a dramatic drop in activity compared to the normal version, both in live cells and in lab-based experiments. Interestingly, the mutant enzyme still folded correctly and was located in the right part of the cell, which ruled out structural instability as the cause. This finding made it clear that the PA14 domain plays an essential role in helping the enzyme do its job by stabilizing the active site and enabling key glycan modifications.

The authors wanted to go further and understand how B4GALNT3 works on different substrates. They tested the enzyme’s ability to produce LacdiNAc on a variety of molecules, including N-glycans, O-GalNAc glycans, and glycoproteins. By using advanced biochemical assays and reverse-phase HPLC to analyze reaction products, they found that the normal enzyme efficiently modified these substrates, while the mutant enzyme with no PA14 domain was almost completely inactive. This provided strong evidence that the PA14 domain is not just important but absolutely necessary for B4GALNT3 to function. The team also looked at how LacdiNAc synthesis influences other glycan modifications. They found that when LacdiNAc was present, it inhibited several downstream processes like sialylation, fucosylation, and the synthesis of the HNK-1 epitope. For instance, enzymes such as ST6GAL1 and FUT2, which are involved in adding terminal modifications, were less effective when LacdiNAc was part of the substrate. Through careful in vitro experiments, the researchers confirmed that LacdiNAc alters how enzymes interact with substrates, effectively shaping the overall glycosylation pattern of glycoproteins. The researchers also explored how LacdiNAc interacts with another glycan modification called bisecting GlcNAc, which is added by GnT-III and discovered a reciprocal relationship: bisecting GlcNAc prevented the formation of LacdiNAc, and LacdiNAc partially blocked GnT-III activity. This interaction highlighted the complex regulatory network of glycosylation, showing how different modifications can influence one another.

In conclusion, the new study, led by Professor Yasuhiko Kizuka and colleagues, offered better understanding of glycosylation, with special focus on the creation and regulatory role of LacdiNAc. The practical implications of this research are exciting. For instance, LacdiNAc’s ability to regulate how long proteins stay active in the bloodstream has clear potential for therapeutic applications. Moreover, adding LacdiNAc to therapeutic proteins could help fine-tune their activity or reduce unwanted immune responses. Alternatively, blocking the activity of B4GALNT3 could extend the half-life of certain proteins which make them more effective as treatments. The authors’ findings also have new applications for bone health because with the examination on how LacdiNAc affects proteins like sclerostin which regulates bone mass, the authors has paved the way for potential new treatments for osteoporosis and other bone-related conditions. Furthermore, the link between LacdiNAc and stem cell behavior opens doors for regenerative medicine and cancer therapies, especially in cancers where altered glycan structures play a role in tumor growth and spread. According to the authors and from a structural biology perspective, the role of the PA14 domain in glycosylation deserves further exploration because understanding how it works at a three-dimensional level could lead to advances in enzyme design which will help researchers develop targeted inhibitors or engineered enzymes for medical and research purposes.