岡 昌吾

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.

Nonsulfated Human Natural Killer-1 (nsHNK-1) glycan is a unique trisaccharide structure terminating in glucuronic acid and synthesized by glucuronyltransferase GlcAT-S. This glycan is specifically expressed in the mouse kidney, particularly in proximal tubules and the thick ascending limb of Henle's loop. Renal nsHNK-1 glycan exhibits an age-dependent increase, indicating that its expression is strictly regulated. However, previous studies have primarily focused on male kidneys, leaving potential sex differences unexplored. In this study, we found that renal nsHNK-1 glycan expression is significantly higher in female mice compared with male mice. Notably, no sex differences were observed in the expression of N-acetyllactosamine structures, the substrate for glucuronic acid modification, or in GlcAT-S expression levels. Moreover, analysis of knockout mice for GlcAT-P, an isoform of GlcAT-S, confirmed that GlcAT-P also does not contribute to the sex differences in nsHNK-1 glycan expression. These findings prompted us to investigate the intracellular availability of uridine diphosphate glucuronic acid (UDP-GlcA), the donor substrate for GlcAT-S, as a possible contributor to sex-specific renal nsHNK-1 glycan expression. To investigate this hypothesis, we developed a quantitative ELISA to measure intracellular UDP-GlcA levels. In vivo, wild-type female mice exhibited lower renal UDP-GlcA levels compared with males. However, this difference was abolished in GlcAT-S knockout mice, suggesting enhanced UDP-GlcA consumption in female mice. In HK-2 cells, derived from human proximal tubular epithelium, cultivation under high-glucose conditions elevated intracellular UDP-GlcA, resulting in increased nsHNK-1 glycan expression. Conversely, stimulation of UDP-GlcA consumption via glucuronidation using 4-methylumbelliferone suppressed the high-glucose-induced increase in nsHNK-1 glycan expression levels. Taken together, these findings identify UDP-GlcA availability as a key determinant of nsHNK-1 glycan biosynthesis in the kidney, highlighting a novel regulatory mechanism that contributes to sex-specific glycan expression.

Immunogenicity is a major caveat of protein therapeutics. In particular, the long-term administration of protein therapeutic agents leads to the generation of antidrug antibodies (ADAs), which reduce drug efficacy while eliciting adverse events. One promising solution to this issue is the use of mirror-image proteins consisting of d-amino acids, which are resistant to proteolytic degradation in immune cells. We have recently reported the chemical synthesis of the enantiomeric form of the variable domain of the antibody heavy chain (d-VHH). However, identifying mirror-image antibodies capable of binding to natural ligands remains challenging. In this study, we developed a novel screening platform to identify a d-VHH specific for vascular endothelial growth factor A (VEGF-A). We performed mirror-image screening of two newly constructed synthetic VHH libraries displayed on T7 phage and identified VHH sequences that effectively bound to the mirror-image VEGF-A target (d-VEGF-A). We subsequently synthesized a d-VHH candidate that preferentially bound the native VEGF-A (l-VEGF-A) with submicromolar affinity. Furthermore, immunization studies in mice demonstrated that this d-VHH elicited no ADAs, unlike its corresponding l-VHH. Our findings highlight the utility of this novel d-VHH screening platform in the development of protein therapeutics exhibiting both reduced immunogenicity and improved efficacy.

T7 phage libraries displaying random peptides are powerful tools for screening peptide sequences that bind to various target molecules. The T7 phage system has the advantage of less biased peptide distribution compared to the M13 phage system. However, the construction of T7 phage DNA is challenging due to its long 36 kb linear DNA. Furthermore, the diversity of the libraries depends strongly on the efficiency of commercially available packaging extracts. To address these issues, we examined the combination of seamless cloning with cell-free translation systems. Seamless cloning technologies have been widely used to construct short circular plasmid DNA, and several recent studies showed that cell-free translation can achieve more diverse phage packaging. In this study, we combined these techniques to construct four libraries (CX7C, CX9C, CX11C and CX13C) with different random regions lengths. The libraries thus obtained all showed diversity > 109 plaque forming units (pfu). Evaluating our libraries with an anti-FLAG monoclonal antibody yielded the correct epitope sequence. The results indicate that our libraries are useful for screening peptide epitopes against antibodies. These findings suggest that our system can efficiently construct T7 phage libraries with greater diversity than previous systems.