1. Expression of human salivary histatin and cystatin/histatin chimeric cDNAs in Escherichia coli
L A Bobek, H Tsai, M J Levine Crit Rev Oral Biol Med. 1993;4(3-4):581-90. doi: 10.1177/10454411930040034501.
We have previously constructed recombinants encoding the full-length and truncated forms of cystatin-SN and expressed these in the Escherichia coli expression system pGEX-2T, which expresses foreign sequences as fusion proteins with glutathione S-transferase (GST). Recombinant cystatins were produced and purified in large quantities. The full-length recombinant cystatin-SN exhibited comparable biological activity and secondary structure to natural cystatin, validating the use of the full-length and mutant recombinant proteins for structure-function studies of salivary molecules. In this study, we have expressed histatin-1 cDNA in the pGEX-3X vector and cystatin-SN/histatin-1 or cystatin-SN/histatin-3 chimeric cDNAs in the pGEX-2T vector. Gene splicing by overlap extension (SOE), a PCR-based method, was used for generating the chimeric cDNAs. Each construct was analyzed by DNA sequencing, which showed the correct junctions and reading frames between the GST/histatin-1 and the GST/cystatin/histatin cDNAs. Expression of histatin and cystatin/histatin chimeras was induced by IPTG and the production of the fusion proteins monitored by SDS-PAGE/Coomassie blue staining and in the case of the GST/cystatin/histatin fusion proteins, also by Western blot using anti-cystatin antibody. The results of these studies showed that we have successfully constructed recombinants encoding the individual and chimeric salivary molecules and efficiently expressed these in E. coli expression system pGEX. Purification and characterization of recombinant histatin and cystatin-histatin hybrid proteins are presently ongoing.
2. The efficacy of salivary Histatin-1 protein in wound closure of nicotine treated human periodontal ligament fibroblast cells - In vitro study
Amal Arab, K G Aghila Rani, Roa T Altell, Asmaa A Ismail, Sausan Alkawas, A R Samsudin Arch Oral Biol. 2022 Sep;141:105486. doi: 10.1016/j.archoralbio.2022.105486. Epub 2022 Jun 17.
Objectives: The aims of this study were to investigate the efficacy of Histatin-1 in wound closure as well as effects on gene expression of nicotine-treated human Periodontal Ligament Fibroblast cells (HPDL) in vitro. Design: HPDL grown in 2.5% culture medium treated with 10 ng/ml Histatin - 1 in the presence/absence of 0.5 µM nicotine were subjected to wound assay and migration was studied at 0 h, 6 h, 12 h and 24 h. Cells grown in 2.5% medium served as control. Cell migration was studied by wound gap and transwell migration assays. The effect of Histatin-1 on expression of matrix metalloproteinase 8 (MMP-8), insulin-like growth factor 1 (IGF-1), transforming growth factor beta (TGF-β), collagen type I (COL1) and plasminogen activator inhibitor 1 (PAI-1) were studied. Results: Histatin-1 treatment significantly decreased percentage wound gap at 12 h (62.96 ± 3.22 vs 79.23 ± 1.73; p < 0.05) and at 24 h (38.78 ± 7.59 vs 75.21 ± 4.94; p < 0.001) compared with controls. In nicotine+Histatin-1 treated cells, wound gap decreased to 70.2 ± 2.9% (p < 0.01) at 24 h compared to nicotine alone in which 82 ± 1.64% of wound gap was retained. Transwell migration assays showed significant migration of HPDL with Histatin-1 (p < 0.05). Gene expression demonstrated significant upregulation for IGF-1, TGF β, COL1 and PAI-1 with Histatin-1. Conclusion: Histatin-1 significantly mitigated the effect of nicotine in wound healing assay involving HPDL fibroblast cells at 24 h. Histatin-1 aided wound closure is attributed to the upregulation of IGF-1, TGF β, COL1, and PAI-1 genes.
3. Human Salivary Histatin-1-Functionalized Gelatin Methacrylate Hydrogels Promote the Regeneration of Cartilage and Subchondral Bone in Temporomandibular Joints
Changjing Shi, Yu Yao, Lei Wang, Ping Sun, Jianying Feng, Gang Wu Pharmaceuticals (Basel). 2021 May 19;14(5):484. doi: 10.3390/ph14050484.
The avascular structure and lack of regenerative cells make the repair of osteochondral defects in the temporomandibular joint (TMJ) highly challenging in the clinic. To provide a viable treatment option, we developed a methacrylated gelatin (Gel-MA) hydrogel functionalized with human salivary histatin-1 (Hst1). Gel-MA is highly biocompatible, biodegradable, and cost-effective. Hst1 is capable of activating a series of cell activities, such as adhesion, migration, differentiation, and angiogenesis. To evaluate the efficacy of Hst1/Gel-MA, critical-size osteochondral defects (3 mm in diameter and 3 mm in depth) of TMJ in New Zealand white rabbits were surgically created and randomly assigned to one of the three treatment groups: (1) control (no filling material); (2) Gel-MA hydrogel; (3) Hst1/Gel-MA hydrogel. Samples were retrieved 1, 2, and 4 weeks post-surgery and subjected to gross examination and a series of histomorphometric and immunological analyses. In comparison with the control and Gel-MA alone groups, Hst1/Gel-MA hydrogel was associated with significantly higher International Cartilage Repair Society score, modified O'Driscoll score, area percentages of newly formed bone, cartilage, collagen fiber, and glycosaminoglycan, and expression of collagen II and aggrecan. In conclusion, Hst1/Gel-MA hydrogels significantly enhance bone and cartilage regeneration, thus bearing promising application potential for repairing osteochondral defects.
