Although neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8) and ubiquitin share the highest level of sequence identity and structural similarity among several known ubiquitin-like proteins, their conjugation to a protein leads to distinct biological consequences. In the study, we first identified the NEDD8 protein of Chlamydomonas reinhardtii (CrNEDD8) and discovered that CrNEDD8 is fused at the C-terminus of a ubiquitin moiety (CrUb) in a head-to-tail arrangement. This CrUb-CrNEDD8 protein was termed CrRUB1 (related to ubiquitin 1) by analogy with a similar protein in Arabidopsis thaliana (AtRUB1). Since there is high sequence identity in comparison to the corresponding human proteins (97% for ubiquitin and 84% for NEDD8), a His-CrRUB1-glutathione S-transferase (GST) fusion construct was adopted as the alternative substrate to characterize the specificity of NEDD8-specific peptidase SENP8 for CrNEDD8. The data showed that SENP8 only cleaved the peptide bond beyond the di-glycine motif of CrNEDD8 and His-RUB1 was subsequently generated, confirming that SENP8 has exquisite specificity for CrNEDD8 but not CrUb. To further determine the basis of this specificity, site-directed mutagenesis at earlier reported putative molecular determinants of NEDD8 specific recognition by SENP8 was performed. We found that a single N51E mutation of CrNEDD8 completely inhibited its hydrolysis by SENP8. Conversely, a single E51N mutation of CrUb enabled this ubiquitin mutant to undergo hydrolysis by SENP8, revealing that a single residue difference at the position 51 contributes substantially to the substrate selectivity of SENP8. Moreover, the E51N/R72A double mutant of the CrUb subdomain can further increase the efficiency of cleavage by SENP8, indicating that the residue at position 72 is also important in substrate recognition. The E51N or R72A mutation of CrUb also inhibited the hydrolysis of CrUb by ubiquitin-specific peptidase USP2. However, USP2 cannot cleave the N51E/A72R double mutant of the CrNEDD8 subdomain, suggesting that USP2 requires additional recognition sites.

Ubiquitin (Ub) shares the highest sequence identity with neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8) in the Ub-like protein family. However, different enzyme systems are precisely employed for targeting Ub and NEDD8 to specific substrates. The molecular determinants for distinguishing between Ub and NEDD8 by Ub-specific peptidases (USPs) remain poorly characterized. By replacing the non-conserved residues of Ub with their NEDD8 equivalents by mutagenesis, and vice versa, we observed that the Ub4K, Ub12E, and Ub14E mutants partially and the Ub4K/12E/14E/72A mutant completely prevented their hydrolysis by USP2. The NEDD84F and NEDD814T mutants were slightly hydrolyzed by USP2; however, the NEDD812T/14T/72R and NEDD84F/12T/14T/72R mutants were accessible for hydrolysis by USP2, suggesting that Ub and NEDD8 residues 4, 12, 14, and 72 serve as the molecular determinants for specific recognition by USP2. We also demonstrated that the level of inhibition caused by Ub mutants with multiple mutation sites was not purely additive when compared with the single mutation results. Furthermore, USP2 was determined to bind to the N-terminus of Ub to form a stable interaction, after which it binds with the C-terminus of Ub to ensure substrate specificity. The same results were also discovered when Ub, Ub4K/12E/14E/72A, NEDD8, and NEDD84F/12T/14T/72R were incubated with USP21.

Therapeutics reducing bone turnover, such as denosumab (Dmab), an anti-RANKL antibody, can provide treatments for patients with bone destruction. However, some patients with osteoporosis or localized primary bone tumors and many patients with various types of bone-metastatic cancer display unsatisfactory responses to Dmab. For achieving greater efficiency of RANKL neutralization in the bone microenvironment by enhancing the distribution of Dmab to the bone, we reengineered Dmab by fusing with single-chain variable fragments of an antibody specific for osteonectin (On), which is abundantly expressed in osseous tissues. The bispecific antibody, Dmab-FvOn, showed a similar activity as Dmab in inhibiting RANKL as examined in an osteoclast differentiation assay. When administered to mice, Dmab-FvOn was found to localize in increased proportions at the endosteum of the bone where osteonectin is abundant. Our study suggests that by linking anti-RANKL with an osteonectin-targeting moiety, a greater proportion of the therapeutic effector can be distributed in the bone. Future studies are needed to investigate whether the bispecific antibody can achieve higher therapeutic efficacy and lower toxicity.

In allergic patients sensitive to house dust mite allergens, the inhaled allergenic proteins are dissolved in mucosal fluid, cross mucosal epithelia of the airway, and induce crosslinking of FcεRI-bound allergen-specific IgE on mast cells and basophils, leading to the degranulation of those inflammatory cells. Our group has proposed that allergen-specific IgG, delivered to the airway mucosa, can bind to the incoming allergens and preclude their entry into the mucosal layer. In this work, we prepared human monoclonal antibodies by isolating dust mite allergen-specific single B cells from patients with atopic dermatitis and performing PCR on the VH and VL cDNA of the B cells. One mAb, 1D11, an IgG1 (γ1, λ), bound strongly to both Der p 2 and Der f 2 and recognized a linear epitope in the peptide segment, a.a. residues #41-70, of the two proteins. We proceeded evaluating the ability of 1D11 to trap Der p 2 and Der f 2 in a culture of rat basophilic leukemic (RBL) SX-38 cells, which were charged with allergen-specific IgE contained in patient sera. In the culture, 1D11 was capable of inhibiting the degranulation of RBL SX-38 cells triggered by Der p 2 and Der f 2. Furthermore, the activation was reduced to the background level when protein A-conjugated Sepharose was employed to remove the 1D11-allergen complexes. These results indicate that mAb 1D11 is effective in trapping Der p 2 and Der f 2 allergens and prevent their activation of allergen-specific IgE-sensitized basophils.