Collagen is a fundamental component of the extracellular matrix, providing structural support, and playing a critical role in the integrity and functionality of various tissues. Among the different types of collagens, collagen type III is particularly notable for its presence in tissues that exhibit elastic properties, such as the skin, blood vessels, and internal organs (1). Constituting approximately 5-20% of the total collagen content in the human body, collagen type III is the second most abundant collagen after type I. It is essential for the proper assembly and stability of collagen fibrils, while its absence leads to significant structural abnormalities (2, 3). Studies on knock-out mice of the collagen type III gene (COL3A1) reveal irregularly sized collagen fibers in the skin dermis and the aortic adventitia, underscoring the importance of collagen type III in regulating collagen fiber size (3). The physiological importance of COL3A1 is further underscored by its association with Ehlers–Danlos syndrome (EDS) IV, a severe genetic disorder that affects the connective tissues (4). Mutations in COL3A1 lead to compromised structural integrity in various organs (4, 5), highlighting its critical role in maintaining tissue strength and elasticity.

Skin aging is influenced by both intrinsic factors, such as genetics and hormones, and extrinsic factors, including chronic exposure to UV radiation and environmental pollutants (6-9). These factors concurrently lead to histopathological changes and diminished collagen synthesis, resulting in reduced skin elasticity and the appearance of wrinkles (10-12). Collagen and elastin play essential roles in maintaining the structural integrity and elasticity of the skin within the dermis (9, 13). Collagen, a fibrous protein crucial for preserving the skin’s mechanical strength, predominates in the dermis (14). The collagen molecule consists of three polypeptides known as alpha chains, which are characterized by higher concentrations of glycine, proline, hydroxyproline, and alanine. This distinctive amino acid composition is intricately linked to collagen formation, which is associated with fibrogenesis (15). While collagen type I has been extensively studied for its role in skin health (16, 17), the role of collagen type III remains less explored.

This study aims to address this gap by developing and purifying recombinant proteins derived from human collagen type III. By focusing on the central triple helical domain (THR) of collagen type III alpha 1 chain, we generated functional fragments that can enhance collagen synthesis, promote cell proliferation, support wound healing, and penetrate the dermal layer of the skin. These functional fragments offer potential applications in cosmetic and therapeutic products aimed at combating skin aging and damage.

Development and purification of functional recombinant hCOL3A1 fragment proteins

The hCOL3A1 protein consists of several distinct domains that contribute to its function (18). These include signal peptide, which directs the nascent protein to the endoplasmic reticulum for further processing and secretion, and the N–terminal and C–terminal propeptide domains (procollagen III amino terminal propeptide segment, PIIINP; procollagen III carboxy terminal propeptide segment, PIIICP). These propeptide domains are involved in the proper assembly of collagen triple helices and are cleaved off during maturation (19). The central triple helical domain (THR), characterized by the repeating Gly–X–Y sequence, is essential for the stability and tensile strength of collagen fibrils (20). Additionally, the short non-helical telopeptide domains at both ends of the triple helix play a crucial role in the cross-linking of collagen molecules, contributing to the formation of strong and stable collagen fibers (20, 21). These domains work together to ensure the proper structure, stability, and function of hCOL3A1, which is integral to the structural integrity of various tissues (Fig. 1A).

To verify the physiological activity and utility of hCOL3A1 and facilitate its various applications, we aimed to obtain recombinant proteins. However, studies using a bacterial expression system revealed that the expression efficiency of the wild type hCOL3A1 was very low (data not shown). Based on these results and our previous research, we shifted our development focus from the full-length wild type hCOL3A1 to protein fragments utilizing the essential domains of hCOL3A1. This approach reduces the size of the recombinant protein, increases expression efficiency, and allows specific peptides to be obtained that display activity.

To develop various collagen peptides derived from human hCOL3A1, we cloned the wild-type hCOL3A1 and various fragment proteins into the pET−28a vector through PCR. These constructs were then expressed in E. coli Rosetta2 (DE3) strain, and purified as previously described (16, 17). We specifically targeted the THR domain, essential for collagen activity. The expression of hCOL3A1−THR−M and THR−C was confirmed (Fig. 1B). Further division of these regions into smaller fragments and subsequent testing for expression led to the successful detection of the protein expression of hCOL3A1−THR−M1 and hCOL3A1−THR−M4. However, expression of the remaining hCOL3A1-derived fragment proteins could not be detected (Fig. 1C and 1D, and Supplemental Fig. 1). The expression and quality of the purified hCOL3A1−THR−M1 and THR−M4 proteins produced using the bacterial system were confirmed through electrophoresis and Coomassie blue staining. Finally, mass spectrometry analysis was used to verify their identity (Fig. 1E).

Effect of collagen biosynthesis by hCOL3A1−THR−M1 and hCOL3A1−THR−M4

Given that hCOL3A1 functions in conjunction with collagen type I, we examined the effect of hCOL3A1−THR−M1 and hCOL3A1−THR−M4 on collagen type I expression in skin cells. Plasmids encoding an empty vector (control), hCOL3A1−THR, hCOL3A1−THR−M1, and hCOL3A1−THR−M4 were transfected in HDF cells, and their expressions were confirmed by western blot (Fig. 2A). The amount of procollagen type I, a precursor of collagen type I, was measured by ELISA. As shown in Fig. 2B, a significant increase in the production of procollagen type I was observed with hCOL3A1−THR−M1 and hCOL3A1−THR−M4. Although this increase was not as high as that induced by TGF–β1 (10 ng/ml), it was approximately 1.6-fold higher than the control. Interestingly, hCOL3A1−THR had no effect on collagen synthesis.

Next, we tested the potential of skin cells to produce collagen type I by treating them with purified recombinant proteins hCOL3A1−THR−M1 and hCOL3A1−THR−M4. The purified proteins were confirmed by western blot and Coomassie blue staining (Fig. 2C). When HDF cells were treated with hCOL3A1−THR−M1 and hCOL3A1−THR−M4 for 48 hours, and compared with TGF–β1-treated conditions, the expression of procollagen type I increased in proportion to the treatment concentration of the hCOL3A1-derived fragment proteins. Although this increase was still lower compared to the TGF–β1-treated group, it was approximately 1.3 to 2-fold higher than the control (Fig. 2D). Additionally, we found that hCOL3A1−THR−M1 and COL3A1−THR−M4 were almost equally effective, whether treated with intracellularly expressed or purified proteins (Fig. 2B and 2D).

Effects of recombinant hCOL3A1−THR−M1 and hCOL3A1−THR−M4 proteins on cell proliferation and wound healing

TGF–β1 and EGF have previously been reported to promote skin cell proliferation (11, 22). Therefore, we compared their effects on cell proliferation with those of hCOL3A1−THR−M1 and hCOL3A1−THR−M4. Primary human dermal fibroblasts (HDF) and human keratinocyte (HaCaT) cells were treated with TGF–β1 (10 ng/ml), EGF (1 μg/ml), and hCOL3A1−THR−M1 or hCOL3A1−THR−M4 (1 or 10 μg/ml) for 24 hours. As shown in Fig. 3A and 3B, treatment of HDF cells with COL3A1−THR−M1 or COL3A1−THR−M4 resulted in a significant increase in cell proliferation, comparable to the effects of TGF–β1 or EGF. Additionally, HaCaT cells exhibited a similar response, with even more pronounced reactivity. These findings indicate that hCOL3A1−THR−M1 and hCOL3A1−THR−M4 can enhance the proliferation of skin cells.

Next, we examined the effects of hCOL3A1−THR−M1 and hCOL3A1−THR−M4 on wound healing. To evaluate these effects, we used HDF and HaCaT cells, which are commonly utilized as in vitro model of skin wound healing assays (16, 17). We tested the effects of non-treated control, TGF–β1 (10 ng/ml), EGF (1 μg/ml), and hCOL3A1−THR−M1 or hCOL3A1−THR−M4 (1 or 10 μg/ml) on a scratch-wound healing assay using HDF (Fig. 3C and 3E) and HaCaT (Fig. 3D and 3F) cells for 16 or 20 hours. Treatment of HDF and HaCaT cells with recombinant hCOL3A1 proteins (hCOL3A1−THR−M1 or hCOL3A1−THR−M4) resulted in cell migration that was almost equivalent to that observed in the TGF–β1 and EGF treated groups. Therefore, hCOL3A1−THR−M1 and hCOL3A1−THR−M4 are expected to possess the ability to repair skin damage caused by aging or injury, by enhancing the proliferation and migration of human skin cells.

Effect of skin tissue permeation by recombinant hCOL3A1 proteins

Using KeraSkin–FT, a full-thickness skin model derived from normal human keratinocytes and fibroblast, we verified the skin permeability of COL3A1−THR−M1 and COL3A1−THR−M4 in a multilayer structure environment similar to the human epidermis and dermis (Biosolution Co., Ltd., Korea). As shown in Figure 4, after 3 hours of treatment, hCOL3A1−THR−M1, but not hCOL3A1−THR−M4 (data not shown), reached the stratum corneum of the epidermis, and after 9 hours of treatment, penetrated the dermis layer (9, 24, and 48 hours). These results suggest that the hCOL3A1−THR−M1, but not hCOL3A1−THR−M4, protein can penetrate the dermal layer of the skin, inducing various enhancing effects on skin cells, and thus demonstrating the potential to inhibit the cellular aging of skin.

In conclusion, hCOL3A1-derived functional proteins, hCOL3A1−THR−M1 and hCOL3A1−THR−M4, enhance collagen type I synthesis, cell proliferation, and wound healing. Furthermore, hCOL3A1−THR−M1 is able to permeate through the skin layers and reach the dermal layer. Therefore, the functional proteins derived from hCOL3A1, specifically hCOL3A1−THR−M1 (referred to as HUCOLLATIN3), have demonstrated the ability to protect against or ameliorate the potential skin damage caused by aging and various environmental factors. This suggests their potential use as functional ingredients in cosmetics and cellular therapies. However, further validation is needed to assess their cytotoxicity and immune responses in clinical settings.

Antibodies and reagents

The following antibodies were obtained from each supplier: anti−α−tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-HA and anti-His (Cell Signaling Technology, Danvers, MA, USA). The following reagents were used: Epidermal Growth Factor (EGF, PeproTech−ThermoFisher Scientific), transforming growth factor-beta 1 (TGF−β1, R&D Systems, Minneapolis, MN, USA), dimethyl sulfoxide and WST−1 (Sigma−Aldrich, St. Louis, MO, USA).

Cell culture

Primary human dermal fibroblasts (HDFs; American Type Culture Collection, Manassas, VA, USA) and immortalized human keratinocytes HaCaT cells (Addexbio, San Diego, CA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Slough, UK) containing 4.5 g/L glucose, 2 mM L-glutamine (Sigma-Aldrich), 100 IU/ml penicillin, 100 μg/ml streptomycin (Sigma-Aldrich), and 10% (v/v) fetal bovine serum (FBS; ThermoFisher Scientific). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

Construction of expression vectors for hCOL3A1 and fragments

All cDNA inserts of hCOL3A1 were generated by PCR using Q5 Hot Start High−Fidelity DNA Polymerase and corresponding primers: hCOL3A1−1−forward primer (Fw) 5’-AAAAAGCTTGCATGATGAGCTTTGTGC-3’, hCOL3A1−153−reverse primer (Rv) 5’-GAAGCGGCCGCGGGAGAATAGTTCTGA-3’, hCOL3A1−154−Fw 5’-ACTAAGCTTGCCAGTATGATTCATATG-3’, hCOL3A1−1221−Rv 5’-ATTGCGGCCGCTCCATAATACGGGGCA-3’, hCOL3A1−1222−Fw 5’-CGTAAGCTTGCGATGAACCAATGGATT-3’, hCOL3A1−1464−Rv 5’-AGTGCGGCCGCTAAAAAGCAAACAGGG-3’, hCOL3A1−168−Fw 5’-TAGAAGCTTGCGGACTCGCAGGCTATC-3’, hCOL3A1−1196−Rv 5’-ACAGCGGCCGCGCAAGGACCAGGGGCA-3’, hCOL3A1−426−Fw 5’-ATGAAGCTTGCGGACTGCGAGGTGGTG-3’, hCOL3A1−683−Rv 5’-CCAGCGGCCGCACGTTCACCAGGGGCA-3’, hCOL3A1−684−Fw 5’-CTGAAGCTTGCGGACCTCCTGGATTGG-3’, hCOL3A1−941−Rv 5’-CCTGCGGCCGCTGGTGGGCCCTGGGCA-3’, hCOL3A1−560−Rv 5’- CCTGCGGCCGCACTTTCTCCTTGACTT-3’, hCOL3A1−555−Fw 5’- CCCGAATTCGGAAGTCAAGGAGAAAGT-3’, hCOL3A1−624−Fw 5’- CCCGAATTCGGGCCTGGTGGTGACAAA-3’, hCOL3A1−758−Rv 5’- CCCGCGGCCGCATCTTTCCCTGGGACA-3’, hCOL3A1−738−Fw 5’- GGAGAATTCGGTCCAAAGGGTGACAAG-3’, hCOL3A1−872−Rv 5’- CCAGCGGCCGCAGCAGCACCAGGTCCA-3’, hCOL3A1−807−Fw 5’- GGCGAATTCGGACCTGCTGGTTTCCCT-3’, hCOL3A1−684−Fw 5’-CTGAAGCTTGCGGACCTCCTGGATTGG-3’, hCOL3A1−1196−Rv 5’-ACAGCGGCCGCGCAAGGACCAGGGGCA-3’, hCOL3A1−813−Fw 5’- CTGAAGCTTGCGGTGCTCCTGGACAGA-3’, hCOL3A1−1070−Rv 5’- CCTGCGGCCGCAGCAGGGCCACTTTCT-3’, hCOL3A1−942−Fw 5’- CTGAAGCTTGCGGAGCTCCAGGCCCAC-3’, and hCOL3A1−941−Rw 5’- CCTGCGGCCGCTGGTGGGCCCTGGGCA-3’

Then, cDNA inserts encoding hCOL3A1 full−length (FL, 1-1464 aa) and hCOL3A1 fragments were cloned into pET−28a or pcDNA3.1−HA and resulting plasmids were verified by Sanger sequencing. hCOL3A1 fragments included hCOL3A1−THR (168-1196 aa), hCOL3A1−THR−N (168−683 aa), hCOL3A1−THR−M (426−941 aa), hCOL3A1−THR−C (684-1196 aa), hCOL3A1−THR−M1 (426−560 aa), hCOL3A1−THR−M2 (555−683 aa), hCOL3A1−THR−M3 (624−758 aa), hCOL3A1−THR−M4 (738-872 aa), hCOL3A1−THR−M5 (807−941 aa), hCOL3A1−THR−C1 (684-941 aa), hCOL3A1−THR−C2 (813−1070 aa), and hCOL3A1−THR−C3 (942-1196 aa).

Expression and purification of the hCOL3A1 recombinant proteins

pET−28a constructs containing hCOL3A1 FL and fragments were transformed into the E. coli Rosetta2 (DE3). Following protein induction using IPTG, bacterial pellets were harvested and lysed through three rounds of sonication (10 sec duration with a 50 sec interval). Crude extracts were cleared by centrifugation at 4°C and 14,000 rpm for 30 min. His-tagged proteins were purified using Ni−NTA resin (GE Healthcare) at 4°C for 1 hour with rotation. Resin bound proteins were washed with ice−cold washing buffer [phosphate-buffered saline (PBS) containing 50 or 100 mM imidazole, pH 7.4] and eluted with ice-cold elution buffer (PBS containing 500 mM imidazole, pH 7.4). Eluates were dialyzed at 4°C overnight against PBS (pH 7.4) in a dialysis tube (Sigma-Aldrich). The purified proteins were stored at −80°C until use.

Cell viability assay

HDF or HaCaT cells were seeded at concentration of 3 × 10³ cells or 5 × 10³ cells per well in 100 μl of culture medium into 96 well plates and incubated at 37°C in a humidified atmosphere containing 5% CO₂. After 24 hours, the culture media were replaced with fresh DMEM containing 0.1% FBS and treaeted with TGF−β1 (10 ng/ml), EGF (1 μg/ml), or hCOL3A1 (1 or 10 μg/ml) for 24 hours. Cell viability was measured by WST-1 Assay. 10 μl of WST−1 reagent (Sigma-Aldrich) was directly added to each well and incubated for 4 hours at 37°C in a humidified atmosphere containing 5% CO₂. After 1 min of shaking, the absorbance quantification of the samples, the formazan dye produced by metabolically active cells, was measured using microplate reader against a background control as blank. The wavelength for measuring the absorbance of the formazan product is 440 nm (between 420-480 nm). The results were obtained through at least three independent experiments.

Measurement of collagen biosynthesis

HDF or HaCaT cells were seeded at a concentration of 3 × 10³ cells per well in 100 μl of culture medium into 96 well plates and incubated at 37°C in a humidified atmosphere containing 5% CO₂. After 24 h, the culture media were replaced with fresh DMEM containing 0.1% FBS and treated with TGF−β1 (10 ng/ml), EGF (1 μg/ml), or hCOL3A1 as indicated (1, 10 or 100 μg/ml) for 48 hours. To analyze the collagen production (procollagen type I), the conditioned media were collected from each well and clarified by centrifugation at 3,000 rpm for 15 min. The concentration of biosynthesized and secreted procollagen type I in conditioned media was measured using an ELISA kit (Takara Bio Inc., Otsu, Japan) according to the manufacturer’s protocol. The results were obtained through at least three independent experiments.

Transfection and western blotting

HDFs were transfected with pcDNA3.1−HA vectors (empty, hCOL3A1−THR, hCOL3A1−THR−M1 or hCOL3A1−THR-M4) using the Lipofectamine 3000 Transfection Reagent (ThermoFisher Scientific). Cells were harvested at 48 hours post-transfection and were lysed in RIPA buffer [50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% sodium dexoycholate, 0.1% sodium dodecyl sulfate, 1.5 mM Na₃VO₄, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM β−glycerolphosphate, and EDTA free protease inhibitor cocktail (Roche)]. Lysates were cleared by centrifugation at 14,000 rpm for 15 min at 4°C. Protein samples were separated by SDS−PAGE and transferred to polyvinyl difluoride (PVDF) membrane (GE Healthcare Amersham). Membranes were blocked in TBS with 5% non-fat milk and 0.1% Tween 20 and probed with primary antibodies. Secondary antibodies horseradish-peroxidase-conjugated were purchased from ThermoFisher Scientific and ECL reagent (GE Healthcare Amersham) was used for detection.

LC/MS and MS/MS data analysis

The protein bands in the SDS-PAGE gel were subjected to trypsin digestion and prepared for liquid chromatography-mass spectrometry (LC-MS) analysis, following a previously described protocol (23). The samples were analyzed using an LTQ−Orbitrap Velos (ThermoFisher Scientific) connected to an Easy-nano LC II system (ThermoFisher Scientific) incorporated with an autosampler. Acquired data was analyzed using Sequest (XCorr Only; version v.27, rev. 11) and X! Tandem [version CYCLONE (2010.12.01.1)] using the UniProt human database.

In vitro wound-healing assay

The scratch wound-healing assay was previously described (16, 17, 24). In brief, cells were seeded at a concentration of 1.5 × 10⁵ cells per well into 24 well plates and incubated until confluent at 37°C in a humidified atmosphere containing 5% CO₂. The cell monolayer was gently scratched across the center of the well using a sterile 200 μl pipette tip. Following scratching, the medium was aspirated, and cells were washed twice with PBS. Fresh culture medium containing 0.1% FBS was then added, and the cells were treated with TGF−β1 (10 ng/ml), EGF (1 μg/ml) or hCOL3A1 as indicated (1 or 10 μg/ml) for 16 hours. To quantify wound closure, images of the same fields were captured immediately after scratching (0 hour) and after 16 hours using a microscope. Image analysis was performed using ImageJ by manually selecting the total area of the wound regions. The experiments were performed triplicate, and two fields were analyzed for each replicate (n = 6).

Skin tissue permeation assay

This experiment was conducted according to the guidelines for in vitro skin absorption tests provided by the Ministry of Food and Drug Safety of the Republic of Korea. For the percutaneous absorption assessment, FITC-labeled COL3A1 (COL3A1−FITC) or vehicle was applied to each treatment condition on the artificial skin model. After the application, any remaining sample on the surface was removed with DPBS, and cryo-sectioning was performed. To identify the boundary between the epidermis and dermis of the tissue, DAPI staining was used, and the permeated COL3A1 was visualized through FITC.

Statistical analysis

Results in graphs are expressed as mean ± SD or SEM as indicated in figure legends, for the indicated number of observations. Statistical significance was determined by the Student’s t-test (two-tailed, unequal variance) using GraphPad Prism software package (GraphPad Inc.). P-value < .05 is considered significant and is indicated in figure legends.

This work was supported by an INHA UNIVERSITY Research Grant to K.K.

The authors have no conflicting interests.