Lumican is a major small leucine-rich proteoglycan (SLRP) in Atlantic cod (Gadus morhua L.) skeletal muscle

Abstract

Knowledge on fish matrix biology is important to ensure optimal fish -quality, -growth and -health in aquaculture. The aquaculture industry face major challenges related to matrix biology, such as inflammations and malformations. Atlantic cod skeletal muscle was investigated for collagen I, decorin, biglycan, and lumican expression and distribution by real-time PCR, immunohistochemical staining and Western blotting. Immunohistochemical staining and Western immunoblotting were also performed using antibodies against glycosaminoglycan side chains of these proteoglycans, in addition to fibromodulin. Real-time PCR showed highest mRNA expression of lumican and collagen I. Collagen I and proteoglycan immunohistochemical staining revealed distinct thread-like structures in the myocommata, with the exception of fibromodulin, which stained in dense structures embedded in the myocommata. Chondroitinase AC-generated epitopes stained more limited than cABC-generated epitopes, indicating a stronger presence of dermatan sulfate than chondroitin sulfate in cod muscle. Lumican and keratan sulfate distribution patterns were strong and ubiquitous in endomysia and myocommata. Western blots revealed similar SLRPs sizes in cod as are known from mammals. Staining of chondroitin/dermatan sulfate epitopes in Western blots were similar in molecular size to those of decorin and biglycan, whereas staining of keratan sulfate epitopes coincided with expected molecular sizes of lumican and fibromodulin. In conclusion, lumican was a major proteoglycan in cod muscle with ubiquitous distribution overlapping with keratan sulfate. Other leucine-rich proteoglycans were also present in cod muscle, and Western blot using antibodies developed for mammalian species showed cross reactivity with fish, demonstrating similar structures and molecular weights as in mammals.

Small leucine-rich proteoglycans in the vertebrae of Atlantic salmon Salmo salar

“…In mammals, SLRPs have been detected in osteoblast and hypertrophic chondrocytes of limb long bones and in developing bone matrices (Bianco et al 1990, Takagi et al 2000, Alini & Roughley 2001, Yang et al 2012 and appear to play a role related to establishing ossification fronts and matrix organization. All 3 proteins showed a molecular size between 40 and 50 kDa, which is in accordance with results from other teleosts (Tingbø et al 2012) and mammals (Iozzo 1999, Pedersen et al 2001) reflecting cross-species similarities. We found that SLRPs in Atlantic salmon osteoblasts are increasingly transcribed during differentiation in vitro, as described in mammalian osteoblast cell lines , Mochida et al 2003, Waddington et al 2003, Chen et al 2005, Parisuthiman et al 2005.…”

“…The nitrocellulose membrane was then probed with the polyclonal antibodies decorin (ab35378), lumican (ab70191) and biglycan (ab20033) (Abcam) diluted 1:5000 in Tris-buffered saline (TBS) added to 0.5% teleostan gelatine. These antibodies are specific to teleosts (Tingbø et al 2012). Alkaline phosphatise (AP) conjugated secondary antibodies (anti-mouse IgG(H+L), Promega) diluted 1:7500 were then applied to the membranes.…”

Lumican deficiency results in cardiomyocyte hypertrophy with altered collagen assembly

“…Previous studies using mouse models of LUM deficiency have determined its requirement for normal collagen fiber assembly in the skeletal muscle, [14] tendon [15] and cornea [16, 17]. We evaluated the spatiotemporal localization of LUM throughout cardiac development and using a LUM deficient mouse model determine a potentially critical role for LUM in murine cardiac development.…”

Sulphated glycosaminoglycans and proteoglycans in the developing vertebral column of juvenile Atlantic salmon (Salmo salar)

“…In a recent study, we immunolocalized lumican in the chordocytes of Atlantic salmon (Pedersen et al 2013 ). Lumican is reported to carry KS also in teleosts such as Atlantic cod (Tingbo et al 2012 ) and zebrafish (Souza et al 2007 ).…”