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인체�유사�콜라겐�타입 I 알파 �

• INCI : Hydrolyzed Collagen

• M.W. : 분자량: ��kDa 이하
• 주름개선�효과
• 콜라겐�합성�효능�탁월

• 피부�세포�침투�효능
• 세포�복원�효능
• ELASTIN, FIBRONECTIN 증가�효과
Accessions
• 피부�밀도�개선�효과 Description MW(Da) Coverage(%)
• 비건�인증
COL1A1 COL1A1 12151 93.3

Protein sequence coverage: 100%

Accessions Description MW(Da) Coverage(%)

COL1A2 COL1A2 12400 100

Accessions Description MW(Da) Coverage(%)
%0% BMB Rep. 2021; 54(6): 329-334
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Reports COL3A1 12194 97.8
COL3A1

Effects of human collagen α-1 type I-derived proteins on

collagen synthesis and elastin production in human dermal
fibroblasts

1,#
2,#
3,4
3,4
5
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Su Jin Hwang , Su Hwan Kim , Woo-Young Seo , Yelin Jeong , Min Cheol Shin , Dongryeol Ryu , Sang Bae Lee ,
Young Jin Choi 2,7,8, * & KyeongJin Kim 3,4, *
2
1 ABIOTECH Co., Ltd., Suwon 16675, Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Department of
3
4
Biomedical Sciences, College of Medicine, Inha University, Incheon 22212, Program in Biomedical Science & Engineering, Inha University,
Effects of hCOL1A1-derived peptides on skin cells
Su Jin Hwang, et al.
6
5
Incheon 22212, Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 16419, Division of Life
Effects of hCOL1A1-derived peptides on skin cells
Su Jin Hwang, et al.
factors (FGF), hepatocyte growth factor (HGF), transforming
ABIOTECH Co., Ltd., Suwon 16675,
7
growth factor-β 1 (TGF-β1), epidermal growth factor (EGF), plate-
Sciences, Jeonbuk National University, Jeonju 54896, Center for Food and Bioconvergence, Seoul National University, Seoul 08826,
let-derived growth factor (PDGF), and insulin-like growth factor
Effects of hCOL1A1-derived peptides on skin cells
(IGF) are directly or indirectly associated with HDF motility
Su Jin Hwang, et al.
minal region spanning amino acid residues 743-880 of SMA
(12-14). The impact of these growth factors on HDFs covers a
8 Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea Effects of recombinant hCOL1A1-CED protein on cell
and overlapping with both SMC and SMD is the functional
proliferation and collagen biosynthesis
broad range of biological phenotypes, such as cellular pro-
We compared the effects of hCOL1A1-CED and EGF on HDF
and effective region of hCOL1A1 that affects collagen type I
liferation, regeneration, and metabolism (15-17).
Department of Anatomy and Cell Biology, type I (hCOL1A2) promotes collagen synthesis, elastin produc- cell proliferation, as these have previously been reported to
synthesis. Then, the SME was further designated as hCOL1A1-
Our previous study has shown that human collagen alpha-2
promote cell proliferation (8, 18, 23). As shown in Fig. 3A,
collagen effective domain (hCOL1A1-CED).
both hCOL1A1-CED protein (10 µg/ml) and EGF (1 µg/ml)
tion, and wound healing in normal dermal fibroblasts (18).
Isolation and identification of recombinant hCOL1A1-CED
Our analysis has identified that the hCOL1A2-derived peptide
protein
been reported to significantly increase collagen type I synthesis
promotes the improvement of skin properties compared with showed similar HDF cell proliferation (Fig. 3A). TGF-β1 has
Next we cloned a histidine (His)-tagged hCOL1A1-CED into
and proliferation in both embryonic pulmonary fibroblasts and
the effects of marine collagen. Although human collagen type I
Biomedical Institute for Convergence at SKKU is composed of a triple helix of human collagen alpha-1 type I HDFs in a crowded cell culture system (8, 24). Therefore, to
the pET-28a (His-tag) vector. Recombinant proteins were pro-
further investigate the effects of hCOL1A1-CED, we compared
duced by the Escherichia coli Rosetta2 (DE3) strain and purified
(hCOL1A1) wound together with hCOL1A2 (11), the effects of
as previously represented (22). To ensure the molecular com-
hCOL1A1 on skin properties have not been investigated. In
its effect with that of TGF-β1 or EGF on collagen synthesis. We
position ratio, purified hCOL1A1-CED was isolated and visual-
determined the amount of collagen type I secreted in the
this study, we found that hCOL1A1-derived proteins promote
ized as a single band of approximately 21 kDa by SDS-PAGE,
culture media of HDFs stimulated with TGF-β1 (10 ng/ml),
collagen synthesis, elastin production, and cell migration in
Collagen type I is the most abundant form of collagen in human fluenced by environment and lifestyle factors, such as chronic
EGF (1 µg/ml), or hCOL1A1-CED (10 µg/ml). We found that
as also shown the uninduced or induced TCP (total cell proteins)
HDFs and HaCaT keratinocytes.
(BICS), Sungkyunkwan University School of
(Fig. 2A). Then, hCOL1A1-CED was confirmed using an anti-His
hCOL1A1-CED (10 µg/ml), TGF-β1 (10 ng/ml) and EGF (1 µg/
antibody (Fig. 2B) as well as an anti-COL1A1 (CED-specific)
RESULTS AND DISCUSSION
ml) showed similar increases in collagen type I synthesis
induction, endogenous Col1a1 gene expression and fibroblast
antibody (Fig. 2C).
tissues, and is composed of two identical α-1 type I chains and light exposure and chemicals (2). These factors incorporate
cell proliferation (Fig. 3B-D).
Identification of the efficiency of hCOL1A1 domains in
To further confirm the sequences of purified recombinant
hCOL1A1-CED, we analyzed the peptides from in-gel digestion
collagen type I synthesis
Furthermore, hCOL1A1-CED was detectable in HDF cell lysates
with trypsin using liquid chromatography-mass spectrometry
even after the culture medium treated with hCOL1A1-CED
Medicine, Suwon 16419, Department of
Previous studies have suggested that collagen type I synthesis
(LC/MS), showing a representative MS/MS spectrum for the
was washed out (Fig. 3E), suggesting that hCOL1A1-CED is
and fibroblast cell proliferation are required to maintain the
identified peptides originating from hCOL1A1 ( 781 GESGPSGPA
an α-2 type I chain organized in a triple helical structure. A histopathological and immunohistochemical alternations in
strength and elasticity of the skin (19-21). We have previously
bioactive, similar to other hydrolyzed collagen peptides, and
can be absorbed and utilized by HDFs. Together, these results
GPTGAR 796 , 1297.61724 m/z) in Fig. 2D and Supplementary
reported that hCOL1A2-derived proteins promote collagen
type I synthesis, elastin production, and fibroblast cell prolife-
demonstrate that hCOL1A1-CED significantly increases collagen
Table 1.
type I synthesis and fibroblast proliferation.
ration in normal HDFs (18). To investigate the biological func-
tion of hCOL1A1 another major component of human collagen
previous study has shown that human collagen α-2 type I each skin layer, such as changes in skin appearance after UV
Biomedical Sciences, College of Medicine, Inha
Fig. 3. hCOL1A1-CED stimulates cell proliferation and collagen synthesis in HDFs. (A) HDF cells were treated with EGF (0.1 and 1 µg/ml) and
type I, in human skin fibroblasts we examined the effects of
Fig. 1. Identification of hCOL1A1 domain for the synthesis of collagen
hCOL1A1-CED (1 and 10 µg/ml) for 48 h. Cell proliferation, induced by EGF and hCOL1A1-CED, was determined by 3-(4,5-dimethylthiazol-
type I. (A) Schematic representing full-length hCOL1A1 and its various
hCOL1A1 domains on collagen type I synthesis in normal
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (B and C) HDF cells were treated with EGF (1 µg/ml; 166.7 nM), hCOL1A1-CED (10 µg/ml;
deletion mutants. (B and C) Human dermal fibroblasts (HDFs)
HDFs, similar to our previous study (18). Full-length hCOL1A1
840.9 nM), or TGF-β1 (10 ng/ml; 781.3 pM) for 48 h. The amount of collagen type I in the HDF culture media and mRNA levels of
were transfected with plasmids encoding hemagglutinin (HA), as
(hCOL1A2) promotes collagen synthesis, wound healing, and exposure (3-5). Furthermore, both intrinsic and extrinsic factors
Col1a1 in HDF cells were measured by enzyme-linked immunosorbent assay (B) or qRT-PCR (C), respectively. Cell proliferation was determined
and various deletion mutants were generated (Fig. 1A), and
the negative control (NC), HA-tagged full length hCOL1A2 (FL),
by MTT assay (D). (E) HDF cells were treated with EGF (1 µg/ml), TGF-β1 (10 ng/ml), or hCOL1A1-CED (1 and 10 µg/ml), for 48 h. Cells
were collected 48 h post-treatment and cell lysates were analyzed by immunoblotting with anti-His (upper panel) or anti-α-tubulin antibodies,
and each of the deletion mutants (N-terminal chain [N], middle
Normal HDFs were transfected with plasmids encoding hemag-
University, Incheon 22212, Program in
glutinin (HA) (negative control; NC), HA-tagged full-length
respectively. Results are presented as the mean ± standard deviation of three independent experiments. Student’s t-test was used for statistical
chain [M], or C-terminal chain [C]). Forty-eight hours after transfec-
analyses (*P ƕ 0.05, **P ƕ 0.005). cell lysates were analyzed by
tion, cells were collected and
COL1A1 (FL; chain domain), and the deletion mutants, namely
immunoblotting with anti-HA and anti-tubulin antibodies (B). The
elastin production in normal human dermal fibroblasts (HDFs). result to decreased levels of elastin and collagen synthesis in
of the N-terminal (N), middle (M), and C-terminal chain (C).
amount of collagen type I in transfected HDF culture media was
The expression of hCOL1A1 FL and deletion mutants was
measured with an enzyme-linked immunosorbent assay (ELISA) kit
Effects of recombinant hCOL1A1-CED protein on migration
CED (10 µg/ml). Elastin synthesis significantly increased in the
(C). (D and E) HDF cells were transfected with plasmids encoding
analyzed by immunoblotting (Fig. 1B), and the levels of colla-
hCOL1A1-CED-treated group, compared with the TGF-β1 (10
HA (NC) and each
and elastin production of the indicated mutants (small middle A
gen type I were measured using an hCOL1A1 enzyme-linked
[SMA], small middle B [SMB], small middle C [SMC], small
Biomedical Science & Engineering, Inha
Previous studies have determined that the abundant amino
ng/ml) and EGF (1 µg/ml)-treated groups and the NC group,
However, the biological effects of human collagen α-1 type I fibroblasts and increased melanin production in melanocytes
immunosorbent assay (ELISA) kit. Interestingly, the M deletion
middle D [SMD], or small middle E [SME]). Forty-eight hours after
acid residues in collagen peptides support cellular growth and
whereas fibronectin synthesis was not affected by hCOL1A1-CED
mutant significantly induced collagen type I synthesis compared
transfection, cells were collected and cell lysates were analyzed
treatment (Fig. 4C, D), likely representing that the lack of
by immunoblotting with anti-HA and anti-tubulin antibodies (D).
proliferation (25). We further investigated the effects of hCOL
with the NC or other deletion mutants (Fig. 1C).
1A1-CED on cell adhesion and growth using the human kera- kit
binding affinity to fibronectin (29) does not speculate on fibro-
The amount of collagen type I was measured with an ELISA
In order to narrow down the protein region in the M-domain
in a transfected HDF-cultured media (E). Results are presented as
(hCOL1A1) on various skin properties have not been investi- (6-8). critical for collagen type I synthesis, we further constructed tinocyte cell line HaCaT, which is an in vitro model of skin nectin synthesis.
mean ± SD of three independent experiments. Student’s t-test
Next, to test whether hCOL1A1-CED is associated with skin
wound healing (26). We tested the effects of TGF-β1, EGF, or
several deletion mutants inside the M-domain. HDFs were
was used for statistical analyses (*P ƕ 0.05, **P ƕ 0.005).
hCOL1A1-CED in the transwell migration assay using HaCaT
aging, we further measured melanin synthesis. We observed
University, Incheon 22212, Division of Life
transfected with plasmids encoding HA (NC) and the deletion
that melanin synthesis was not altered by treatment with
cell monolayers and the scratch-wound healing assay using
mutants, namely small middle A (SMA), small middle B (SMB),
HaCaT and HDF cells, and as shown in Fig. 4A, B, hCOL1A1-
hCOL1A1-CED in B16F10 melanoma cells, whereas vitamin C
gated. Here, we isolate and identify the hCOL1A1-collagen Collagen and elastin are structural proteins in the skin that
deletion mutant significantly increased collagen type I synthesis
small middle C (SMC), small middle D (SMD), and small
treatment significantly reduced melanin synthesis (Fig. 4E).
CED treatment significantly increased cell migration, compared
compared with the other deletion mutants; however, the other
middle E (SME). Cells were analyzed after 48 h of transfection
Additionally, the gene expression of matrix metalloproteinase
using immunoblotting (Fig. 1D) and the amount of collagen
with the NC.
mutants (SMA, SMB, SMC, or SMD) were also capable of
1 (Mmp1), Mmp2, Mmp3, or tissue inhibitor of metalloproteinase
Previous studies have suggested that the skin aging is induced
type I was measured with an hCOL1A1 ELISA kit. The SME
increasing collagen type I synthesis (Fig. 1E). Thus, the C-ter-
Sciences, Jeonbuk National University, Jeonju
effective domain (CED) which promotes collagen type I syn- maintain both elasticity and firmness (9). Interestingly, the fibrous
by intrinsic and extrinsic pathway, which are both accompanied
1 (Timp1) was significantly downregulated by hCOL1A1-CED
Fig. 2. Identification of purified hCOL1A1-CED. (A) Coomassie Blue staining of uninduced (without IPTG), induced TCP (total cell protein),
treatment in HDF cells (Fig. 4F), which is consistent with
with histopathological and immunohistochemical alternations
or isolated hCOL1A1-CED showed a prominent band; arrows indicate that hCOL1A1-CED was identified using (B) anti-His and (C) anti-
330 %0% Reports
hCOL1A1 antibodies by western blot analysis. (D) Identification of hCOL1A1-CED protein in the gel band
previous results that these changes are associated with aging
(1). Intrinsic pathway of skin aging is a process of both the of isolated hCOL1A1-CED by liquid
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chromatography-mass spectrometry. The panels show the representative MS/MS spectrum for the identified peptides of GESGPSGPAGPTGAR
qualitative and quantitative changes, including diminished or
and collagen degradation (30). Together, these results demon-
(1297.61724 m/z, 2+).
strate that hCOL1A1-CED induces cell migration and elastin
defective collagen and elastin production in the dermis (27,
thesis. Recombinant hCOL1A1-CED effectively induces cell protein collagen constitutes the majority of the skin and con-
synthesis, but not fibronectin synthesis.
28). To evaluate the effect of hCOL1A1-CED on elastin syn-
54896, Korea
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In conclusion, hCOL1A1-derived CED enhances collagen
thesis, we analyzed elastin levels in the culture media of HDFs
type I and elastin synthesis, cell proliferation as well as cell
treated with TGF-β1 (10 ng/ml), EGF (1 µg/ml), or hCOL1A1-
proliferation and collagen biosynthesis in HDFs, as well as tributes to maintain mechanical strength of tissues (10). The
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332 %0% Reports
increased cell migration and elastin production. Based on collagen molecule is composed of three chains wound toge-
these results, hCOL1A1-CED may be explored further for its ther in a tight triple helix. Every third amino acid is glycine
potential use as a preventative agent against skin aging. [BMB that fits perfectly inside the helix. The triple helix is mainly
Reports 2021; 54(6): 329-334] composed of glycine, followed by proline (or hydroxyproline),
and then alanine residues (11). Fibroblasts that produce both
collagen and the elastin matrix can be induced by glycolic
INTRODUCTION acid, and these distinctive fibroblast cells are observed in the
papillary level of the second layer of the dermis (9). Together,
Skin aging is a common dermatological problem and is subject collagen has a specific amino acid composition, which is
to intrinsic and extrinsic processes (1). Intrinsic skin aging is a further associated with fibrogenesis.
physiological change, chronologically influenced by genetic Collagen type I, the most abundant type of collagen in humans,
and hormonal factors, whereas skin aging is extrinsically in- is a major structural protein dominant in the bone (more than
90% of the organic mass), tendons, ligaments, cornea, and
many interstitial connective tissues (11). Different types of
*Corresponding authors. Young Jin Choi, Tel: +82-2-880-4851; Fax: collagen vary in the primary amino acid sequences of their
+82-2-873-5695; E-mail: choiyj@snu.ac.kr; KyeongJin Kim, Tel: +82- polypeptide chains. Structurally, type I collagen comprises two
32-860-9870; Fax: +82-32-885-8302; E-mail: kimkj@inha.ac.kr identical α-1 type I chains and an α-2 type I chain organized
#
These authors contributed equally to this work. in a triple helix (11).
https://doi.org/10.5483/BMBRep.2021.54.6.038 Migration of human dermal fibroblasts (HDFs) is essential for
skin wound healing and remodeling. The proliferation of HDFs
Received 9 March 2021, Revised 22 March 2021, causes their migration into the wound bed, leading to expres-
Accepted 26 April 2021 sion of thick actin bundles in myofibroblasts and the synthesis
of new extracellular matrix (ECM) (12). Previous studies have
Keywords: Collagen synthesis, Elastin, hCOL1A1, Human dermal identified that growth factors/cytokines such as fibroblast growth
fibroblasts
ISSN: 1976-670X (electronic edition)
Copyright ; 2021 by the The Korean Society for Biochemistry and Molecular Biology
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