岡 昌吾

N-glycolylneuraminic acid (Neu5Gc), one of the two major sialic acid species in mammals, cannot be biosynthesized in humans because of the inactivation of the responsible enzyme, CMP-Neu5Ac hydroxylase (CMAH). Consequently, Neu5Gc is recognized as an immunologically foreign molecule, resulting in anti-Neu5Gc antibodies production in humans. Meanwhile, dietary Neu5Gc can be metabolically incorporated into glycans and expressed on tissues. Neu5Gc therefore functions as a xeno-autoantigen in humans. The co-existence of Neu5Gc-containing molecules in tissues and circulating anti-Neu5Gc antibodies is thought to drive human-specific chronic inflammation termed xenosialitis and may contribute to vascular disease such as atherosclerosis. Vascular endothelium is a potential major site of Neu5Gc accumulation. Here, we propose the pathological significance of Neu5Gc accumulation likely depends not only on its presence, but also on the glycan structures on which it is displayed. These endothelial Neu5Gc-containing structures remain poorly defined because of the lack of anti-Neu5Gc antibodies to distinguish linkage- and context-specific epitopes. Here, we characterized a vascular Neu5Gc-containing glycan epitope using novel anti-Neu5Gc monoclonal antibodies. Immunohistochemical analyses of mouse tissues showed enriched localization of the epitope for anti-Neu5Gc monoclonal antibody, #23-5, on arterial endothelium. By correlation analysis of glycosyltransferase gene expression and biochemical analyses, the epitope recognized by #23-5 was identified as an α2,8-linked Neu5Gc-containing disialyl structure. In vivo induction of the #23-5 epitope as a xeno-autoantigen was confirmed using Cmah knockout (Cmah-KO) mice after two days of intravenous administration of per-acetylated ManNGc. These findings provide a structural foundation for future studies of Neu5Gc-dependent vascular xenosialitis.

Immunogenicity remains a major challenge in antibody therapeutics. Antibodies can be recognized as foreign by immune cells, leading to the induction of antidrug antibodies that ultimately compromise therapeutic efficacy. This response is primarily driven by proteolytic processing within antigen-presenting cells and the subsequent presentation of peptide fragments to T cells via major histocompatibility complex molecules. As mirror-image proteins are expected to exhibit resistance to proteolytic degradation, we have been developing mirror-image VHH antibodies composed entirely of d-amino acids. Previously, we generated a d-VHH antibody with specific binding activity to vascular endothelial growth factor A (VEGF-A) and demonstrated that it did not induce antidrug IgG antibodies in a mouse model. However, this d-VHH antibody did not inhibit the VEGF-VEGF receptor interaction, and the immunological processes leading to the lack of IgG antibody induction had not been experimentally demonstrated. In this study, we constructed a new phage-displayed library to improve the hit rate and enable the efficient chemical synthesis of d-VHH antibodies. Using this library, mirror-image screening against d-VEGF identified new VHH antibody sequences with specific binding. The synthesized d-VHH antibody inhibited the VEGF-VEGF receptor interaction. Next, we investigated the immunogenicity of d-VHH antibody using a conventional l-VHH antibody as a reference. The d-VHH antibody resisted enzymatic degradation and, in a mouse model, did not induce inflammatory cytokine production, T-cell responses, or the formation of antidrug antibodies against the administered antibody. Collectively, these findings highlight the potential of inhibitory d-VHH antibodies as a promising approach for developing low-immunogenic therapeutic antibodies.

Persistent synaptic plasticity is essential for memory, yet its stabilizing mechanisms remain incompletely understood. AMPA-type glutamate receptors (AMPARs) mediate most fast excitatory transmission in the brain, but how extracellular posttranslational modifications regulate their functional dynamics in vivo remains unclear. Here, we identify a single endogenous N-linked glycan at Asn401 of the AMPAR GluA1 subunit as a regulator that links receptor gating, membrane microdomain organization, and memory persistence. Loss of this glycan markedly reduced AMPAR desensitization and promoted resensitization. Biochemical and imaging analyses showed that GluA1 lacking the N401 glycan preferentially partitions into ganglioside-rich lipid raft microdomains through enhanced ganglioside binding, thereby disrupting activity-dependent receptor trafficking and promoting excessive receptor internalization. Using GluA1 N401Q knock-in mice, we show that loss of this glycan selectively impairs hippocampal long-term potentiation maintenance and compromises contextual and spatial memory persistence, while leaving learning acquisition largely intact. Notably, this site exhibits partial occupancy in the mammalian brain, indicating that it functions as an endogenous tunable constraint on synaptic stability. Together, these findings establish site-specific glycosylation as a mechanism that coordinates AMPAR gating with membrane compartmentalization to control persistent synaptic plasticity and memory in vivo.

Nonsulfated Human Natural Killer-1 (nsHNK-1) glycan is a trisaccharide biosynthesized by β-1,3-glucuronyltransferase 2 (GlcAT-S) and expressed in the mouse kidney, especially the apical membrane of proximal tubules. Although such apical membrane proteins are known to shed into urine, the presence and significance of urinary nsHNK-1 glycans have remained unexplored. Here, we demonstrated that nsHNK-1 glycan is detectable in mouse urine and identified its major carrier as meprin A subunit alpha (MEP1A), a metalloprotease enriched at the apical membrane of proximal tubules and known to be excreted into urine. Mass spectrometry revealed nsHNK-1 glycosylation at four sites on MEP1A, with the highest occupancy at Asn221 in the catalytic domain. In GlcAT-S knockout mice, urinary MEP1A lacked the nsHNK-1 glycan and exhibited reduced enzymatic activity, suggesting nsHNK-1 glycosylation has a functional contribution. Induced tubular injury decreased urinary MEP1A-associated nsHNK-1 levels, following a similar pattern to reduced MEP1A-associated nsHNK-1 levels in the kidney. In contrast, glomerular injury resulted in increased MEP1A-associated nsHNK-1 expression in the kidney, accompanied by elevated urinary nsHNK-1 levels. These findings suggest that urinary nsHNK-1 levels differentially reflect renal dysfunction and may serve as a novel biomarker of kidney function.

Glucuronyltransferase GlcAT-P is a rate-limiting enzyme involved in the biosynthesis of the Human Natural Killer-1 carbohydrate and is essential for acquiring higher brain functions. Alternative splicing produces two isoforms, short-form GlcAT-P (sGlcAT-P) and long-form GlcAT-P (lGlcAT-P), which share identical peptide sequences except for an additional 13 amino acids (AA) in the cytoplasmic N-terminal tail of lGlcAT-P. Although sGlcAT-P localizes to the Golgi apparatus (GA), where many glycosyltransferases reside, lGlcAT-P is distributed in both the GA and endoplasmic reticulum (ER). However, the mechanisms responsible for this distinct intracellular distribution remain poorly understood. In this study, we explored the role of the 13 AA in the cytoplasmic N-tail of lGlcAT-P in trafficking between the GA and the ER using the Retention Using Selective Hooks system. Our findings revealed that lGlcAT-P undergoes enhanced retrograde trafficking from the GA to the ER, whereas its anterograde trafficking from the ER to the GA remains largely unaffected. In addition, three glutamic acid residues within the 13 AA of lGlcAT-P were identified as crucial for promoting retrograde trafficking. These results suggest that the ER distribution of lGlcAT-P is primarily governed by Golgi-to-ER trafficking regulated by specific sequences in its cytoplasmic N-tail.