Serine-/Cysteine-Based sp2-Iminoglycolipids as Novel TLR4 Agonists: Evaluation of Their Adjuvancy and Immunotherapeutic Properties in a Murine Model of Asthma.

Glycolipids with TLR4 agonistic properties can serve either as therapeutic agents or as vaccine adjuvants by stimulating the development of proinflammatory responses. Translating them to the clinical setting is hampered by synthetic difficulties, the lack of stability in biological media, and/or a suboptimal profile of balanced immune mediator secretion. Here, we show that replacement of the sugar fragment by an sp2-iminosugar moiety in a prototypic TLR4 agonist, CCL-34, yields iminoglycolipid analogues that retain or improve their biological activity in vitro and in vivo and can be accessed through scalable protocols with total stereoselectivity. Their adjuvant potential is manifested in their ability to induce the secretion of proinflammatory cytokines, prime the maturation of dendritic cells, and promote the proliferation of CD8+ T cells, pertaining to a Th1-biased profile. Additionally, their therapeutic potential for the treatment of asthma, a Th2-dominated inflammatory pathology, has been confirmed in an ovalbumin-induced airway hyperreactivity mouse model.

DSPP dosage affects tooth development and dentin mineralization.

Dentin Sialoprotein (DSP) and phosphophoryn (PP) are two most dominant non-collagenous proteins in dentin, which are the cleavage products of the DSPP (dentin sialophosphoprotein) precursor protein. The absence of the DSPP gene in DSPP knock-out (KO) mice results in characteristics that are consistent with dentinogenesis imperfecta type III in humans. Symptoms include thin dentin, bigger pulp chamber with frequent pulp exposure as well as abnormal epithelial-mesenchymal interactions, and the appearance of chondrocyte-like cells in dental pulp. To better understand how DSPP influences tooth development and dentin formation, we used a bacterial artificial chromosome transgene construct (BAC-DSPP) that contained the complete DSPP gene and promoter to generate BAC-DSPP transgenic mice directly in a mouse DSPP KO background. Two BAC-DSPP transgenic mouse strains were generated and characterized. DSPP mRNA expression in BAC-DSPP Strain A incisors was similar to that from wild-type (wt) mice. DSPP mRNA expression in BAC-DSPP Strain B animals was only 10% that of wt mice. PP protein content in Strain A incisors was 25% of that found in wt mice, which was sufficient to completely rescue the DSPP KO defect in mineral density, since microCT dentin mineral density analysis in 21-day postnatal animal molars showed essentially identical mineral density in both strain A and wt mice. Strain B mouse incisors, with 5% PP expression, only partially rescued the DSPP KO defect in mineral density, as microCT scans of 21-day postnatal animal molars indicated a reduced dentin mineral density compared to wt mice, though the mineral density was still increased over that of DSPP KO. Furthermore, our findings showed that DSPP dosage in Strain A was sufficient to rescue the DSPP KO defect in terms of epithelial-mesenchymal interactions, odontoblast lineage maintenance, along with normal dentin thickness and normal mineral density while DSPP gene dosage in Strain B only partially rescued the aforementioned DSPP KO defect.

DSP-PP C-Terminal Conservation Is Crucial for Accurate DSP-PP Precursor Cleavage.

Dentin Sialoprotein (DSP) and Phosphophoryn (PP), acidic proteins critical to dentin mineralization, are translated from a single transcript as a DSP-PP precursor that undergoes proteolytic processing to generate DSP and PP. Because of the difficulty in obtaining large amounts of DSP-PP, we used a Sf9-baculovirus expression system to yield large amounts of DSP-PP240 recombinant protein, a variant form of rat DSP-PP. Previous evidence stated that DSP-PP240 produced by baculovirus-infected Sf9 cells can be cleaved accurately into DSP and PP by the endogenous processing enzyme Sf9 Tolloid-Related 1 (TLR1), a homolog for human Bone Morphogenic Protein 1 (BMP1) and the proposed protease to cleave DSP-PP in human. It was also discovered via mass spectrometric analysis that the specific cleavage occurred at the site: SMQG447|D448DPN. In addition, we reported that any mutations within the DSP-PP P4 to P4'cleavage site can block, impair or accelerate DSP-PP cleavage, which suggest that its BMP1 cleavage site is highly conserved to regulate its cleavage efficiency. Furthermore, mutations outside of the DSP-PP P4 to P4' cleavage site can impair or accelerate DSP-PP cleavage. Here, we investigate the role of the highly conserved DSPP C-terminal region in DSP-PP cleavage. We generated a DSP-PP C-terminal mutation by substituting the terminal two aspartate residues for two histamine residues (DD/HH-DSP-PP). To test the impact of the DD/HH mutant on DSP-PP cleavage, we used the Sf9 expression system's endogenous TLR1 and exogenous recombinant BMP1. The DD/HH mutation was shown to block DD/HH-DSP-PP cleavage into DSP and PP by both TLR1 and BMP1 in vitro. Taken together, these evidence supports our hypothesis that the C-terminal peptides D686D687 actively participates in controlling DSP-PP cleavage and that C-terminal conservation is critical for proper DSP-PP precursor cleavage by TLR1 and BMP1.