Characterization of site-specific N-glycosylation signatures of isolated uromodulin from human urine.

Uromodulin (Umod, Tamm-Horsfall protein) is the most abundant urinary N-glycoprotein produced exclusively by the kidney. It can form filaments to antagonize the adhesion of uropathogens. However, the site-specific N-glycosylation signatures of Umod in healthy individuals and patients with IgA nephropathy (IgAN) remain poorly understood due to the lack of suitable isolation and analytical methods. In this study, we first presented a simple and fast method based on diatomaceous earth adsorption to isolate Umod. These isolated glycoproteins were digested by trypsin and/or Glu-C. Intact N-glycopeptides with or without HILIC enrichment were analyzed using our developed EThcD-sceHCD-MS/MS. Based on the optimized workflow, we identified a total of 780 unique intact N-glycopeptides (7 N-glycosites and 152 N-glycan compositions) from healthy individuals. As anticipated, these glycosites exhibited glycoform heterogeneity. Almost all N-glycosites were modified completely by the complex type, except for one N-glycosite (N275), which was nearly entirely occupied by the high-mannose type for mediating Umod's antiadhesive activity. Then, we compared the N-glycosylation of Umod between healthy controls (n = 9) and IgAN patients (n = 9). The N-glycosylation of Umod in IgAN patients will drastically decrease and be lost. Finally, we profiled the most comprehensive site-specific N-glycosylation map of Umod and revealed its alterations in IgAN patients. Our method provides a high-throughput workflow for characterizing the N-glycosylation of Umod, which can aid in understanding its roles in physiology and pathology, as well as serving as a potential diagnostic tool for evolution of renal tubular function.

Snoopligase-catalyzed molecular glue enables efficient generation of hyperoligomerized TRAIL variant with enhanced antitumor effect.

Clinical application of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is predominantly limited by its inefficient apoptosis induction in tumor cells, which might be improved by using molecular superglue-mediated hyperoligomerization to increase its valency. Here, the minimal superglue peptide pairs, including Snoopligase-catalyzed SnoopTagJr/SnoopDogTag and SpyStapler-catalyzed SpyTag/SpyBDTag, were individually fused at the N- or C-terminus of the TRAIL promoter to produce superglue-fusion TRAIL variants. Similar to native trivalent TRAIL, these superglue-fusion TRAIL variants were highly expressed in Escherichia coli (E. coli) and spontaneously trimerized. In the presence of Snoopligase or SpyStapler, the trivalent superglue-fusion TRAIL variants were predominantly crosslinked into hexavalent TRAIL variants. Nevertheless, Snoopligase was more efficient than SpyStapler in the production of hexavalent TRAIL variants. In particular, Snoopligase-catalyzed trivalent TRAIL variants with N-terminal fusion of SnoopTagJr/SnoopDogTag produced hexavalent SnHexaTR with the highest yield (∼70%). The in vitro cytotoxicity of SnHexaTR was 10-40 times greater than that of TRAIL in several tumor cells. In addition, compared to trivalent TRAIL, hexavalent SnHexaTR showed a longer serum half-life and greater tumor uptake, which resulted in eradication of 50% of tumor xenografts of TRAIL-sensitive COLO 205. In mice bearing TRAIL-resistant HT-29 tumor xenografts, hexavalent SnHexaTR combined with bortezomib encapsulated in liposomes also showed robust tumor growth suppression, indicating that hyperoligomerization mediated by minimal molecular superglue significantly increased the cytotoxicity and antitumor effect of TRAIL. As a novel anticancer agent candidate, the hexavalent SnHexaTR has great potential for clinical application in cancer therapy.

A trimeric immunoglobin G-binding domain outperforms recombinant protein G and protein L as a ligand for fragment antigen-binding purification.

Fragment antigen-binding (Fab) has several advantages in the treatment and diagnosis of some diseases. The lack of highly efficient affinity chromatography platform creates a purification bottleneck for the downstream processing of Fab-based products, which raises the urgent need for a novel immunoglobin G (IgG)-binding domain (IgBD) with both high affinity and broad specificity for Fab. SpGC3FabRR (designated CFab) was previously identified as a Fab-selective IgBD, which triggered our interest in evaluating the potential of CFab for Fab purification. However, we found that monomeric CFab showed weak Fab-binding. To increase its affinity, a self-trimerizing domain (tri) was fused to CFab to produce CFab-tri. It was found that CFab-tri existed as a trimer and showed promising binding to Fab derived from IgG of humans, rhesus monkeys, mice, rats, and rabbits. Affinity chromatography demonstrated that the recovery rates of Fab derived from IgG of humans, rats, mice, and rabbits by CFab-tri-HP column were 2- to 5-fold of those by protein G-HP column. Human Fab was effectively purified by both protein L- and CFab-tri-HP column. However, unlike CFab-tri-HP column, protein L-HP column was inefficient for purification of Fab derived from IgG of rats, mice, and rabbits. Notably, rat Fab spiked into the extract of Escherichia coli (E. coli) was effectively recovered by CFab-tri-HP column. These results indicate that CFab-tri outperforms protein G and protein L as a ligand for Fab purification, and CFab-tri-based affinity chromatography might be developed as a novel platform for Fab purification.

Preventive and reparative functions of host-associated probiotics against soybean meal induced growth, immune suppression and gut injury in Japanese seabass (Lateolabraxjaponicus).

A 56-day feeding trial was conducted to examine the preventive and reparative functions of host-associated probiotics against high soybean meal (SM)-induced negative effects in Japanese seabass (Lateolabrax japonicus). Fish continuously fed low SM (containing 16% SM) and high SM (containing 40% SM) diets were named as positive (PC) and negative (C) control, respectively. Preventive functions of probiotics were evaluated by continuously feeding diets LF3 (Lactococcus petauri LF3 supplemented in high SM diet, group PLF3) and LF4 (Bacillus siamensis LF4 supplemented in high SM diet, group PLF4), while reparative functions were estimated by feeding the high SM diet during 0-28 days, then feeding diets LF3 (group RLF3) and LF4 (group RLF4) until day 56. Compared with the group PC, suppressed growth and immunity, and damaged intestinal health were observed in the group C on days 28 and 56. Fish in groups PLF3 and PLF4, rather than in groups RLF3 and RLF4, showed higher growth compared with the group C and displayed similar immune status to the group PC, indicating that the initial and continued application of probiotic LF3 and LF4 can efficiently improve high SM induced growth and immune deficiency in Japanese seabass, but probiotics had limited reparative benefits when they were administrated at the middle of the feeding trial (28 d). Furthermore, probiotics showed good preventive functions and limited reparative functions on gut health via improving intestinal morphology and inflammation markers, for example, decreasing diamine oxidase activity and d-lactate content, while up-regulating anti-inflammatory TGF-ß1 expression and down-regulating pro-inflammatory TNF-α, IL-1ß, and IL-8 expressions. Moreover, dietary supplementation of probiotics (especially on day 56) could effectively shape the gut microbiota, such as significantly decreasing abundances of opportunistic pathogens (phylum Actinobacteria, genera Pseudomonas and Moheibacter on day 28, phylum Proteobacteria, genus Plesiomonas on day 56), significantly increasing gut microbial diversity and abundances of possible beneficial bacteria (phylum Bacteroidetes and genus Lactobacillus on day 28, phyla Firmicutes, Bacteroidetes and Cyanobacteria, genera Bacillus, Lactobacillus and Bacteroides on day 56). In conclusion, we evidenced for the first time that host-associated L. petauri LF3 and B. siamensis LF4 can provide effectively preventive and certain reparative functions against high SM-induced adverse effects in L. japonicus.

A versatile platform for the tumor-targeted delivery of immune checkpoint-blocking immunoglobin G.

Immunotherapies based on immune checkpoint-blocking antibodies have been considered the most attractive cancer treatments in recent years. However, the systemic administration of immune checkpoint-blocking antibodies is limited by low response rates and high risk of inducing immune-related adverse events (irAEs), which might be overcome by the tumor-targeted delivery of these antibodies. To achieve tumor-targeted delivery, immune checkpoint-blocking antibodies are usually modified with tumor-homing ligands through difficult genetic fusion or chemical conjugation. As most immune checkpoint-blocking antibodies are immunoglobin G (IgG) antibodies, we hypothesize that these IgG antibodies might be noncovalently modified with a tumor-homing ligand fused to an IgG-binding domain (IgBD). To test this hypothesis, the tumor-homing ZPDGFRß affibody, which targets platelet-derived growth factor receptor ß (PDGFRß), was fused to the Fab-selective IgBD in a trimeric format. After mixing ZPDGFRß fused to the IgBD with immune checkpoint-blocking IgG against programmed death-ligand 1 (αPD-L1), a novel homogenous complex was formed, indicating that αPD-L1 had been successfully modified with ZPDGFRß fused to the IgBD. ZPDGFRß-modified αPD-L1 bound to both PDGFRß and PD-L1, thus leading to greater tumor uptake and antitumor effects in mice bearing PDGFRß+PD-L1+ tumor grafts. In addition, due to the broad spectrum of IgBD for IgG, immune checkpoint-blocking IgG antibodies against cytotoxic T-lymphocyte-associated protein 4 (αCTLA-4) and signal regulatory protein alpha (αSIRPα) were also modified with ZPDGFRß fused to the IgBD. These results demonstrated that a tumor-homing ligand fused to the IgBD might be developed as a versatile platform for the modification of immune checkpoint-blocking IgG antibodies to achieve tumor-targeted delivery.

A single nucleotide mutation drastically increases the expression of tumor-homing NGR-TNFα in the E. coli M15-pQE30 system by improving gene transcription.

Due to their potent immune stimulation, tumor necrosis factor alpha (TNFα) variants with tumor-homing activity are attractive as novel antitumor drugs. The promising antitumor effect of NGR-TNFα in clinical trials triggered extensive interest in developing novel tumor-homing TNFα variants in recent years. Owing to its promising antitumor effect, NGR-TNFα is usually used as a control for newly developed tumor-homing TNFα variants. In our previous works, we produced a pericyte-targeting Z-TNFα at high levels using the Escherichia coli (E. coli) M15-pQE30 system. To further compare Z-TNFα and NGR-TNFα, we attempted to express NGR-TNFα using the same system. Surprisingly, native NGR-TNFα was expressed at a low (~ 0.2 mg/L) level in E. coli M15 containing the pQE30 plasmid. However, a single nucleotide mutation of C to G, resulting in a substitution of leucine (L) with valine (V) at the start of TNFα, increased the expression of NGR-TNFα by ~ 100 times through improving transcription. In addition, the amino acid substitution showed a little impact on the receptor binding, in vitro cytotoxicity, and in vivo antitumor effect of NGR-TNFα. As fusing NGR to the N-terminus of TNFα with a valine substitution did not reduce the protein yield, the TNFα gene with a C > G mutation might be used to prepare novel tumor-homing TNFα when the native TNFα-based variant is expressed at an extremely low level in E. coli. Notably, in addition to the mutated valine, the impact of N-terminal additional amino acids provided by pQE30 vector on the function of TNFα variant must be carefully evaluated. KEY POINTS : • A single nucleotide mutation increased the expression of NGR-TNFα by two orders. • Nucleotide mutation-induced amino acid substitution did not reduce NGR-TNFα activity.