Presence and Localization of Pro- and Mature Forms of Biglycan and Decorin in Human Costal Cartilage Derived from Chest Wall Deformities

Research Article

Austin J Musculoskelet Disord. 2015;2(1): 1012.

Presence and Localization of Pro-and Mature Forms of Biglycan and Decorin in Human Costal Cartilage Derived from Chest Wall Deformities

Asmar A1, Werner A3, Kelly RE Jr4, Fecteau A5 and Stacey MW1,2*

1Frank Reidy Research Center for Bioelectrics, Old Dominion University, USA

2Department of Pediatrics, Eastern Virginia Medical School, USA

3Department of Pathology, Eastern Virginia Medical School and Med Director of Laboratories, Children's Hospital of The King's Daughters, USA

4Department of Surgery, Eastern Virginia Medical School and Pediatric Surgery Division, Children's Hospital of the King's Daughters, USA

5Hospital for Sick Children, Canada

*Corresponding author: Michael Stacey, Frank Reidy Research Center for Bioelectrics, 4211 Monarch Way, Suite 300, Norfolk, VA23508, USA

Received: November 03, 2014; Accepted: January 07, 2015; Published: February 10, 2015


Costal cartilage is a type of hyaline cartilage that forms rod-like structures that connect the ribs to the sternum. The most common chest wall deformities, pectus excavatum and pectus carinatum involved efective costal cartilage resulting in sternal displacement. Costal cartilage is not widely studied leaving little insight into possible factors involved in the pathogenesis of these pectus deformities. This study focused on the presence and distribution of two important regulators of collagen fibrillogenesis and organization, biglycan and decorin. Immunohistochemical analysis of transverse cross sections of normal and deformed costal cartilage revealed that biglycan and decorin mainly localized in the territorial matrix except for prodecorin which was only found within chondrocytes. Western blot analysis of whole protein extracts demonstrated the presence of both pro and mature forms of biglycan and mature decorin in patients and controls. In normal costal cartilage of different ages, the mature form of decorin was absent in a fetal sample whereas mature biglycan was weakly expressed, suggestive that mature biglycan may play a role in early costal cartilage development. Further studies are needed to determine the functional differences between the pro- and mature forms of biglycan and decorin both in age and disease

Keywords: Decorin; Biglycan; Prodecorin; Probiglycan; SLRP; Chest wall deformities


The biology of costal cartilage is much understudied, yet disorders of costal cartilage have major clinical consequences. Pectus excavatum and pectus carinatum show disfigurement of the chest wall primarily resulting in severely concave or convex chest wall with associated heart, lung, and psychological anomalies. The costal cartilage of patients with chest wall deformities is considered abnormally grown and weak, suggestive of underlying structural defects in growth and assembly. Ultrastructural electron microscopy images have shown that human costal cartilage is unlike other cartilage types. Images appear to show that individual fibers are assembled to collectively form very thick, fascicle structures appearing to consist of large numbers of collagen nanostraws that run parallel along the cartilage length [1]. The morphological form of costal cartilage and how this relates to function is currently unknown.

Small leucine rich proteoglycans (SLRPs) are extra cellular matrix molecules that bind strongly to collagen and other matrix molecules. They are associated with collagen fibril formation and therefore important in the proper formation of ECMs. The cooperation, sequential, timely, orchestrated action of SLRPs that shape architecture and mechanical properties of the collagen matrix and overall importance in disease is reviewed [2,3]. Indeed, SLRP knockout mice exhibit disorganized collagen fibers and loss of connective tissue function [4]. SLRPs are approximately 40kDa, compared to ~200kDa for the large aggregating proteins aggrecan and versican, possess numerous adjacent leucine rich repeats, and one or very few glycosaminoglycan (GAG) side chains. SLRPs are generally expressed in a very tissue-specific manner. Mechanistically, it is accepted that horseshoe-shaped SLRPs interact with collagen molecules by their concave surfaces, and the space inside their curved shape accommodates a single triple helix of collagen [5,6]. Mutations in SLRPs may be important as predisposing genetic factors for diseases of the ECM.

Reports of decreased biomechanical stability of costal cartilage in pectus excavatum patients and a suggested disorderly arrangement and distribution of collagen fibers in chest wall deformities [7,9] led us to examine the size and distribution of two SLRPs expressed in cartilage, decorin and biglycan. Both regulate ECM organization and fibrillogenesis. Interestingly, both sequester transforming growth factor beta (TGFβ), controlling availability of this growth factor and thus growth of cartilage, suggesting a mechanistic role for these two SLRPs in disorders of cartilage growth and assembly [10,11]. Decorin and biglycan are prominent class I members of the SLRP family and are homologous (55% identity at the amino acid level) but with divergent patterns of expression [12]. Despite their similarities, biglycan and decorin have distinct functions which may partially result from differences in GAG chains; decorin having one, and biglycan two O-linked GAG chains respectively and three and two N-linked oligosaccharides[13,14].Biglycan deficiency has been shown to cause spontaneous aortic dissection and rupture in mice [15], a characteristic of Marfan syndrome,a syndrome known to exhibit chest wall deformities. Decorin function is consistent with functions related to fibrillogenesis[2-16].

Decorin and biglycan each occur in two different forms, the pro- and mature (or processed) forms. Proforms of biglycan and decorin have N-terminal pro-peptides that are14 and 21 amino acid residues respectively which are cleaved in the mature forms by bone morphogenic protein (BMP-1) [17,18]. The abundance of proforms of both SLRPs is tissue and age-dependent with the mature form being present in both juvenile and adult tissue and the proform mainly in adult tissue[19,20]. The complex arrangement of fibers observed in costal cartilage and the role of decorin and biglycan in these structures has not been explored. In this study we investigated the presence and distribution of the different forms of decorin and biglycan, hypothesizing that both mature and proforms are functionally required in our late-juvenile samples. This was achieved by immunohistochemistry of costal cartilage from adolescent patients with pectus carinatum and an age-matched control. Our results show the localization of both forms of biglycan and mature form of decorin in the territorial matrix in patient and control samples. Prodecorin was only seen within chondrocytes. Proforms of decorin and biglycan are maintained evolutionarily, suggesting they play an important, but as yet undetermined functional role.

Materials and Methods


Human costal cartilage was obtained from 3 patients with Pectus Carinatum (PC) severe enough to warrant surgical repair. Informed consent was obtained following IRB approval of the protocol at EasternVirginiaMedicalSchool. The IRB protocol currently prevents disclosure of many clinical features, and thus close correlation of clinical phenotype with expression is not possible. A consenting case of pectus excavatum (PE) was obtained from the Hospital for Sick Children, Toronto, Canada. Costal cartilage samples were collected from ribs 6-8 at surgery. Experiments were performed on the round, rod-like, mid-sections of cartilage. All patients were male, with an age range of late-teen to early 20's. Normal costal cartilage was obtained from an age-matched control, a 15 year old male, as well as a 36 week and 81 year oldsample and processed within 24 hours. Resected samples were snap frozen in liquid nitrogen and stored at -80oC until use.

Immunohistochemical analysis

Confirmation of protein distribution was made by immunohistochemistry. Frozen cartilage from 3 cases of PC and an age-matched control were mounted in CRYO-OCTC ompound (Tissue-Tek, CA, USA) and sections (5μm) generated using a Microm HM525 cryostat (Microm International, Walldorf, Germany). Sections were fixed in ice-cold 3:1Methanol:Acetone.Sections were digested with 1.25U/ml Chondroitinase ABC (C3667, Sigma- Aldrich, St. Louis, MO, USA) at 37°C for 2 hours before staining to remove O-linked glyocosaminoglycans and expose the epitope for antibody binding. Blocking, incubation with secondary antibodies, and washes were performed following manufacturer's guidelines for the ImmunoCruz rabbit LSAB staining system (sc-2051, Santa Cruz, Santa Cruz, CA, USA). Tissues were incubated with rabbit polyclonal antibodies specific for probiglycan (LF104), mature biglycan (LF112), prodecorin (LF110), or mature decorin (LF136). Negative controls were produced using normal mouse IgG included in the ImmunoCruz rabbit LSAB staining system.Electronic images were captured using an Olympus DP70 CCD camera through an Olympus BX51 microscope(Olympus America Inc., Center Valley USA). LF-# antibodies were kindly provided by Dr L. Fisher, NIDCR, Bethesda, MD [21].

Protein extraction

Protein extraction was performed using the Fisher-Termine procedure [22] adapted for non-mineralized tissue. All extraction steps were performed with the addition of COMPLETE mini EDTAfree protease inhibitor cocktail (Roche, Mannheim, DE) to prevent the degradation of proteins due to proteases. Transverse segments of whole tissue approximately 0.2g were frozen in liquid nitrogen then crushed into a fine powder. All samples were rinsed with an extraction buffer (4M guanidine-HCl and 50mM Tris, pH 7.5), spun, and the supernatant removed. The samples were then tumbled overnight in a large volume of extraction buffer. The remaining pellets were rinsed twice in extraction buffer before being transferred into Spectra/ Por Dialysis Membranes (Spectrum Labs, Rancho Dominguez, CA, USA), with a 10mm flat width and 12-14kDa molecular weight cut off, and dialyzed in tubes containing extraction buffer for two days. The contents of the dialysis membranes were removed and the supernatants dialyzed against water overnight. The resulting supernatants were then lyophilized.

Western blot analysis

Lyophilized extracts were reconstituted, and approximately 100 μg of each total extractwas separated by electrophoresis on 12.5% Tris-Glycine gels (Bio-Rad, Hercules, CA, USA). Extracts were treated with 1.25 U/ml Chondroitinase ABC (Sigma-Aldrich) at 37°C for 2 hours before loading to remove O-linked glyocosaminoglycans. To investigate cleavage of N-linked deglycosylationfrom core proteins, ABCase treated extracts were denatured at 100°C for 5 minutes then digested with PNGase-F using a N-Glycanase kit (ProZyme, Hayward, CA, USA) for 16 hours at 37°C following manufacturer's guidelines. After electrophoresis, the samples were transferred to nitrocellulose membranes (Bio-Rad). The nitrocellulose membranes were washed, blocked, andincubated with primary and secondary antibodies following manufacturer's guidelines for Odyssey IRDye fluorescent antibodies (LI-COR, Lincoln, NE, USA).Membranes were incubated with rabbit polyclonal antibodies specific for probiglycan (LF104), mature biglycan (LF112), prodecorin (LF110), or mature decorin (LF136). The stained membraneswere imaged using the Odyssey Infrared Imaging System (LI-COR).Positive controls of purified recombinant biglycan and decorin proteoglycans,derived from mouse and human respectively, were kindly provided by Dr M. Young of the NIDCR, Bethesda, MD.


To better understand the function and importance of decorin and biglycan in defective costal cartilage biology, immunohistochemistry and western blot analysis were performed on a limited sample group in order to observe where the protein localizes within the tissue and if any isoforms of the protein exist. The tissue sections and protein extracts were all digested with ABC Chondroitinase to remove O-linked glycosaminoglycans in the N-terminus, a prerequisite to expose the epitope for antibody binding. Examples of patients with defective costal cartilages are shown in (Figure1).