Type III collagen is vital for the formation of normal type I collagen fibrils in the cardiovascular system, intestines, and skin. In the extracellular matrix (ECM), type III collagen is a major component of the interstitial matrix. It is secreted by fibroblasts and other mesenchymal cell types, playing roles in various inflammation-associated pathologies such as lung injury, viral and non-viral liver diseases, kidney fibrosis, hernia, and vascular disorders. Scar tissue contains types I and III collagen with different levels of hydroxylation of lysine and glycosylation of hydroxylysine. During wound healing, the fibrillar collagens, including type III collagen, act as a scaffold for fibroblast attachment. This scaffolding changes the composition of scars, leading to increased scar strength over time. As wound healing continues, this ratio changes to a type I/III ratio of 1:2, which may result in the loss of scar strength. This shift is observed in many conditions such as liver cirrhosis, keloids, and hypertrophic scars due to an increased expression of type III procollagen mRNA (1, 2).
Identifying type III collagen along with type I collagen is generally achieved using ion-exchange chromatography, such as CM-cellulose chromatography under native or reducing conditions. However, this method is only applicable when a large amount of sample is available. In practice, limited sample amounts, such as biopsy specimens, are more common. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is the most common protein analysis method in many laboratories. However, the alpha chains of type I and III collagen show identical Rf values in a Tris buffer system.
Here, we introduce a type III collagen isolation and analysis protocol using a modified PAGE system with urea, which can separate type I and III collagen in a gel. This method is advantageous because it is easy to perform and applicable with small amounts of samples.
Preparation and Analysis of Type III Collagen (3)
First, porcine skins were shaved to remove any hairs. Then, fats in the skin were removed to obtain the dermal section. The dermal sections were chopped into pieces measuring 5 mm x 5 mm. The chopped dermal sections were soaked in 0.05M acetic acid and homogenized by a food mixer. Pepsin was added at a concentration of 0.1 mg/ml and the mixtures were incubated at 4°C overnight. After incubation, the mixture was centrifuged at 10,000 rpm for 20 minutes and the supernatant was collected. The pellet was suspended in 0.05M acetic acid, and these pepsin digestion steps were repeated three times. The collected supernatant was given a 1/10 volume of 1M Tris-HCl, pH 7.5, containing 2M NaCl. The pH was adjusted between 7 and 7.5 using 1M HCl to inactivate pepsin, and the mixture was stored at -20°C. All the supernatants from the three rounds of pepsin digestion were combined.
The combined supernatant was centrifuged at 10,000 rpm for 20 minutes again, and then the supernatant gradually received 33% of its volume of 4M NaCl to achieve a final concentration of 1.2M NaCl. The sample was centrifuged at 10,000 rpm for 20 minutes, and the pellet was saved as the 1.2M precipitate sample. The remaining supernatant gradually received 12% of its volume of 4M NaCl to achieve a final concentration of 1.5M NaCl. The sample was centrifuged at 10,000 rpm for 20 minutes, and the pellet was saved as the 1.5M precipitate sample. The remaining supernatant is the type I collagen fraction.
The 1.2M and 1.5M precipitate samples were dissolved in 0.05M acetic acid. The samples were analyzed by a 5% SDS-PAGE analysis with 4M urea under reducing conditions (4) (Figure 1).
Because both the 1.2M and 1.5M precipitate samples showed almost identical protein patterns, they were combined and dialyzed against 0.1M Tris-HCl buffer, pH 7.5, containing 0.15M NaCl. The samples were filtered with DEAE cellulose to remove pepsin and other contaminated proteins. The flow-through fractions were dialyzed against 0.02M NaH2PO4. The samples were centrifuged at 10,000 rpm for 20 minutes. The pellet was dissolved in 0.05M acetic acid and dialyzed against 0.05M acetic acid. The sample was lyophilized and stored at -20°C as porcine type III collagen.
Figure 1. Porcine type I collagen (I), type III collagen (III), and the 1.2M (1.2M) and 1.5M (1.5M) precipitate samples were dissolved in 0.05M acetic acid and analyzed by 5% SDS-PAGE with 4M urea under reducing conditions.
References
2. J. C. Brown, R. Timpl, The collagen superfamily. Int. Arch. Allergy Immunol. 107, 484-490 (1995).