Differential and restricted expression of novel collagen VI chains in mouse

Matrix Biology 30 (2011) 248–257

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Matrix Biology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m a t b i o

Differential and restricted expression of novel collagen VI chains in mouse Sudheer Kumar Gara a, Paolo Grumati d, Stefano Squarzoni e, Patrizia Sabatelli e, Anna Urciuolo d, Paolo Bonaldo d, Mats Paulsson a,b,c, Raimund Wagener a,b,⁎ a

Center for Biochemistry, Medical Faculty, University of Cologne, 50931 Cologne, Germany Center for Molecular Medicine Cologne (CMMC), University of Cologne, 50931 Cologne, Germany Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50931 Cologne, Germany d Department of Histology, Microbiology & Medical Biotechnologies, University of Padova, 35121 Padova, Italy e Institute of Molecular Genetics (IGM)-CNR, Unit of Bologna c/o IOR, 40136 Bologna, Italy b c

a r t i c l e

i n f o

Article history: Received 19 November 2010 Received in revised form 15 March 2011 Accepted 29 March 2011 Keywords: Collagen VI VWA UCMD Bethlem myopathy

a b s t r a c t Recently, three novel collagen VI chains, α4, α5 and α6, were identified. These are thought to substitute for the collagen VI α3 chain, probably forming α1α2α4, α1α2α5 or α1α2α6 heterotrimers. The expression pattern of the novel chains is so far largely unknown. In the present study, we compared the tissue distribution of the novel collagen VI chains in mouse with that of the α3 chain by immunohistochemistry, immunoelectron microscopy and immunoblots. In contrast to the widely expressed α3 chain, the novel chains show a highly differential, restricted and often complementary expression. The α4 chain is strongly expressed in the intestinal smooth muscle, surrounding the follicles in ovary, and in testis. The α5 chain is present in perimysium and at the neuromuscular junctions in skeletal muscle, in skin, in the kidney glomerulus, in the interfollicular stroma in ovary and in the tunica albuginea of testis. The α6 chain is most abundant in the endomysium and perimysium of skeletal muscle and in myocard. Immunoelectron microscopy of skeletal muscle localized the α6 chain to the reticular lamina of muscle fibers. The highly differential and restricted expression points to the possibility of tissue-specific roles of the novel chains in collagen VI assembly and function. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Collagen VI is an extracellular matrix protein that forms a microfibrillar network in most interstitial connective tissues. It was first isolated from human aortic intima after limited pepsin digestion (Chung et al., 1976) and later from human and bovine placenta (Furuto and Miller, 1980, 1981; Jander et al., 1981, 1983; Odermatt et al., 1983). Collagen VI was long thought to be composed of only three different polypeptide chains α1, α2 and α3. The assembly of collagen VI is a complex multistep process. The three subunits α1, α2 and α3 form a triple helical monomer followed by a staggered assembly into disulfide bonded antiparallel dimers (Knupp and Squire, 2001). Subsequently, the dimers align to form tetramers that are also stabilized by disulfide bonds. The tetramers are secreted into the extracellular matrix to form long molecular chains, known as microfibrils, with a beaded repeat of 105 nm (Furthmayr et al., 1983; Chu et al., 1988). Collagen VI anchors large interstitial structures such as nerves, blood vessels and collagen fibrils into the surrounding connective tissue. Mutations in the genes encoding any ⁎ Corresponding author at: Institute for Biochemistry II, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, D-50931 Cologne, Germany, Tel.: + 49 221 478 6990; fax: + 49 221 478 6977. E-mail address: [email protected] (R. Wagener). 0945-053X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.matbio.2011.03.006

of the collagen VI α1, α2 and α3 chains have been shown to cause Bethlem myopathy (MIM 158810) and Ullrich congenital muscular dystrophy (MIM 254090) in humans (Lampe and Bushby, 2005). Interestingly, in a mouse model it was shown that defective activation of the autophagic machinery is involved in the pathogenic mechanism of these diseases (Grumati et al., 2010). However, recent analysis identified yet three collagen VI chains, α4, α5 and α6 (Fitzgerald et al., 2008; Gara et al., 2008). The genes coding for all three novel chains are arranged in tandem on chromosome 9 in mouse. The novel chains share sequence homology and domain structure with the α3 chain. Each consists of seven von Willebrand factor A (VWA) domains followed by a short collagenous domain, two C-terminal von Willebrand factor A domains, and a unique domain. In addition, the α4 chain carries a Kunitz domain at the C terminus, whereas the α5 chain contains an additional von Willebrand factor A domain and a unique domain. The size of the collagenous domains and the position of the structurally important cysteine residues within these domains are identical between the α3, α4, α5, and α6 chains. Furthermore, the novel chains are completely absent in a collagen VI α1 chain deficient mouse, suggesting that they require the α1 chain for assembly and may substitute for the α3 chain, probably forming α1α2α4, α1α2α5 or α1α2α6 heterotrimers (Gara et al., 2008). In humans, the COL6A4 gene is disrupted by a chromosome break (Fitzgerald et al., 2008; Gara et al., 2008; Wagener

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et al., 2009) as a consequence of a large-scale pericentric inversion on chromosome 3 (Muzny et al., 2006) that converted COL6A4 to the two non-processed pseudogenes COL6A4P1 and COL6A4P2. Interestingly, the 5′ pseudogene (COL6A4P1 or alternatively named DVWA, “double von Willebrand factor A domains”) was recently linked to a predisposition for knee osteoarthritis in an Asian patient cohort (Miyamoto et al., 2008), but this finding could not be confirmed in Caucasians (Valdes et al., 2009). Although COL6A4P1 has the approved status of a pseudogene, a recent study still states that COL6A4P1 transcripts are translated into fragments of the α4 chain without any experimental evidence (Nakajima et al., 2010). Conflicting results exist also on the association of the COL6A5 gene (also named COL29A1) with atopic dermatitis. Whereas COL6A5 was found to be associated in one study (Söderhäll et al., 2007), two others did not show association (Harazin et al., 2010; Naumann et al., 2011) and a fourth showed association of COL6A5 with atopy, but this could not be consistently replicated (Castro-Giner et al., 2009). The expression of the novel collagen VI chains has not yet been studied in detail. RT-PCR showed that both in mouse and human they are more weakly expressed than the α1, α2, and α3 chains that form the “classical” collagen VI (Fitzgerald et al., 2008; Gara et al., 2008). It was shown for human that the α6 chain is expressed in articular cartilage, kidney, skeletal and cardiac muscle, lung, and blood vessels. Lower levels of the α6 chain were found in pancreas and spleen (Fitzgerald et al., 2008). Recently, the α5 and the α6 chains were detected in human dermis and it was shown that their expression can be affected in the collagen VI related diseases Bethlem myopathy and Ullrich congenital muscular dystrophy (Sabatelli et al., 2011). Surprisingly, in another study the human α5 chain was found to be expressed in the epidermis (Söderhäll et al., 2007), where collagen VI has never been found before. For mouse it is only known that the α5 and α6 chains are associated with basement membranes in skeletal muscle, whereas the α4 chain is present in smooth muscle layers of the intestine (Gara et al., 2008). Here we compare the tissue distribution of the novel collagen VI chains in mouse with that of the α3 chain and demonstrate a highly differential and restricted expression. This points to the possibility of tissue-specific roles of the novel chains in collagen VI assembly and function. 2. Results 2.1. Generation of collagen VI α3 chain specific antibodies As antibodies specific for the collagen VI α3 chain were not available, a large fragment consisting of the N-terminal VWA domains N4–N10 from the murine α3 chain was recombinantly expressed in 293EBNA cells to achieve a properly folded protein suited for immunization. After affinity purification, the antibodies specifically detected the α3 chain and did not show cross-reactivity to the new collagen VI chains as determined by ELISA (Supplementary Fig. 1). 2.2. Collagen VI expression in 14.5-day-old embryos Immunostaining for the collagen VI α3 chain of 14.5-day-old embryos showed its broad expression in extracellular matrices (Fig. 1A). In contrast, the new collagen VI chains showed a very restricted expression in only a few tissues. In the bronchi all three new chains are present and partly colocalize with the α3 chain (Fig. 1B–G). Both the α4 chain and the α5 chain are strongly expressed in the epithelial and muscular layers of the bronchial wall, where the α6 chain is only weakly present (Fig. 1B–G). In contrast, the α6 chain shows a specific staining in the parenchyme of the lung, where the α4 and α5 chains are absent (Fig. 1B–G). In the umbilical hernia, the α4 chain is present in the wall of the loops of the midgut, clearly colocalized with the α3 chain (Fig. 1H,I). In contrast, in the inner wall

Fig. 1. Immunohistochemical analysis of a 14.5-day-old mouse embryo. Frozen sections were incubated with affinity purified antibodies against the collagen VI α3 (A, C, E, G, I, K, M, O, Q and S), α4 (B, H and N), α5 (D, J and P) and α6 (F, L and R) chains followed by AlexaFluor 488 (green) and 546 (red) labeled secondary antibodies. The merges of the staining for the α4 (C, I and O), α5 (E, K and Q) and α6 (G, M and S) chains with that for the α3 chain are shown for comparison. A. The collagen VI α3 chain is abundant in nearly all extracellular matrices. Boxes in A show areas in which the new chains are expressed and were studied at higher magnification (B–S). In the bronchi (B–G), the α4 chain and the α5 chain are strongly and the α6 chain is weakly expressed in the epithelial and muscular layer of the bronchial wall. The α6 chain is present in the lung parenchyme (F and G). In the umbilical hernia (L–M) and in the wall of the loops of the midgut, the α4 chain is present and clearly colocalizes with the α3 chain (H–I). In the stomach (N–S), the α4 chain is exclusively expressed in the inner wall (N and O) where the α3 chain is either absent or sparse. Br, Brain; Hr, Heart; Lr, Liver; Vc, Vertebral column; Hl, Hind limb. Scale bar, 100 μm.

of the stomach the α4 chain is exclusively present where the α3 chain is either absent or weakly expressed (Fig. 1N, O). The α5 and α6 chains are absent in hernia and stomach (Fig. 1J–M, P–S).

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2.3. Collagen VI expression in skeletal and cardiac muscles Immunostaining (for summary of results see Table 1) on longitudinal sections of adult mouse skeletal muscle showed that the collagen VI α3 chain is widely expressed in the extracellular matrix surrounding skeletal muscle cells or fibers (Fig. 2A,D,G). It is present in both endomysium and perimysium of muscle cells. The α6 chain is present in similar locations and partially co-localizes with the α3 chain (Fig. 2H,I). The α5 chain showed a strong expression in the perimysium, but was sparse in the endomysium of skeletal muscle fibers (Fig. 2E). It also exhibits a partial co-localization with the α3 chain (Fig. 2F). Interestingly, the α5 and α6 chains are exclusively expressed in some distinct regions (Fig. 2F,I) with the expression of the α6 chain being stronger than that of the α5 chain. In contrast, the α4 chain is completely absent in the skeletal muscle of newborn and adult mouse (Fig. 2B and not shown). The expression of the new chains in the skeletal muscle of newborn mice differs slightly from that in adults (not shown). In newborns the α5 chain shows a weaker expression and the α6 chain is abundant in the endomysium but not in the perimysium and mostly coincides with where the α3 chain is expressed (not shown). In addition, the α5 chain is localized at the perineurium of intramuscular peripheral nerves (not shown). Interestingly, a strong signal was also detected at the basement membrane of neuromuscular junctions as seen by colocalization with α-bungarotoxin (Fig. 2J–L). To study the localization of the novel α5 and α6 chains in murine skeletal muscle at higher resolution, we performed immunoelectron microscopy using specific antibodies against the α5 or α6 chains or against α1,2,3 chain containing collagen VI. However, labeling was obtained only with the α1,2,3 chain and the α6 chain antibodies, probably due to a low efficiency of the α5 chain antibody after preparing specimens for electron microscopy. In the perimysium the α1,2,3 chain (Fig. 3A) and the α6 chain antibodies labeled the microfibrillar network in the vicinity of bundles of fibrillar collagen (Fig. 3B). Some differences in the localization of the α1,2,3 and α6 antibodies were detected in the endomysium, and in particular at the level of basement membranes of muscle fibers and capillary vessels. The α1,2,3 chains were localized at the reticular lamina as well as at the interface of the reticular lamina with the lamina densa of muscle fibers, and also at the basement membrane of capillaries (Fig. 3D,F). In contrast, the α6 chain was restricted to the reticular lamina of muscle fibers (Fig. 3C,E) and it was absent at both the interface with the lamina densa and the basement membrane of capillaries (Fig. 3G). Thus, the electron microscopy results show a differential distribution of the novel α6 chain in the endomysium, which reflects the only partial co-localization with the α3 chain seen with immunofluorescence microscopy. Table 1 Immunohistochemical analysis of the expression of collagen VI chains in adult mice. Tissue

α3

α4

α5

α6

Brain Eye Heart Lung Liver Spleen Intestine Sternum Skeletal muscle Kidney Ovary Testis Skin Blood vessels Vertebral cartilage Knee cartilage

++ ++ ++ ++ ++ ++ ++ ++ +++ ++ ++ ++ ++ ++ ++ ++

– + – – – – +++ – – + +++ ++ + – – –

– (+) + – – – ++ – + ++ ++(+) ++ ++ ++ – –

– ++ ++ – – – – – ++(+) – (+) + – – – –

‘–’ no expression; ‘+’, weak; ‘++’ medium; ‘+++’, strong.

In both newborn and adult mouse hearts, the collagen VI α3 and α6 chains are strongly expressed in the muscle fibers. In contrast, the α5 chain is weakly present in adult and absent in newborn mouse hearts. Similar to skeletal muscle, there is no expression of the α4 chain in mouse myocardium (Supplementary Fig. 2 and not shown). 2.4. Collagen VI expression in intestinal smooth muscle Sections of newborn and adult small intestine, a tissue that contains layers of mucosa and smooth muscle, were stained with the panel of collagen VI specific antibodies (Table 1). The α3 chain is widely expressed in the inner mucosal layer, especially in the villi or papillary mucosal projections towards the lumen, the sub-mucosal connective tissue and the muscular layer of outer longitudinal and inner circular smooth muscle of adult intestine (Fig. 4A,D,G). On the contrary, the α4 chain is completely absent in the muscularis layer but abundant in the sub-mucosal and mucosal layers. It shows strong expression in the lining of villi regions, which occupies the major part of the inner mucosal layer and finely co-localizes with the α3 chain (Fig. 4B,C). The α5 chain showed prominent expression in the mucosal layer and distinctly co-localizes with α3 (Fig. 4E,F). However, it is not as strongly expressed as the α4 chain and exhibits a gradient of decreasing expression towards the lumen. Interestingly, the α6 chain, that is present in both cardiac and skeletal muscles, is absent in the intestinal smooth muscle (Fig. 4H,I). The expression and distribution of the new chains in the intestinal smooth muscle of newborn mouse is similar to that in adult mice (not shown). In addition to the intestinal smooth muscle the α5 chain, but not the α4 and α6 chains, is present in the smooth muscle of the esophagus (not shown). 2.5. Collagen VI chain expression in skin In skin, the novel chains show differences in the expression patterns between newborn and adult stages (Table 1). The α3 chain is widely distributed. It is present at the dermal–epidermal junction, throughout the dermis and also in the subcutaneous muscular layer below the dermis. It is also expressed around the blood vessels and hair follicles, but it is absent in the epidermis (Fig. 4J,M,P). Interestingly, the α5 chain shows a restricted and specific staining surrounding the small blood vessels of the papillary dermis (Fig. 4N), as shown by co-staining with the endothelial cell marker CD31 (Fig. 4T,U). In addition, the α5 chain displays a patchy expression, most probably representing nerves or macrophages in the dermis of adult skin. In all regions it partially co-localizes with the widely expressed α3 chain (Fig. 4O). The α4 chain is very weakly present in the dermal region of adult mouse skin (Fig. 4K). None of the novel chains are found in association with the dermal epidermal basement membrane. The α6 chain is absent in adult skin. However, the expression patterns differ in newborn mice. Newborn skin shows, at the most, weak expression of the novel collagen VI chains whereas the α3 chain is present throughout the dermis and in the basement membranes. Nevertheless, the α6 chain is predominantly present in the muscle layer below the dermis and partially co-localizes with the α3 chain (not shown). Interestingly, the α6 chain, but not the α4 and α5 chains, is present in cornea and lens (Supplementary Fig. 4). 2.6. Collagen VI expression in mouse reproductive organs Another striking differential and selective expression of the novel collagen VI chains can be observed in the reproductive tissues of adult mice, ovary and testis (Fig. 5; Table 1). In sections of mouse ovary the α3 chain is present almost throughout, in parts of both the cortical and medullar regions (Fig. 5A,D,G). The α4 chain is seen in the cortical layer, particularly in the capsule-like structures and in the connective tissue surrounding the follicles, the theca, which also contributes to

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Fig. 2. Immunohistochemical analysis of skeletal muscle from adult mouse. A–I. Frozen sections were incubated with affinity-purified antibodies against the collagen VI α3 (A, D and G), α4 (B), α5 (E) and α6 (H) chains followed by AlexaFluor 488 labeled (green) and 546 (red) secondary antibodies. The merges of the stainings for the α4 (C), α5 (F) and α6 (I) chains with that for the α3 chain are shown for comparison. The α3 and α6 chains are strongly expressed in both endomysial and perimysial connective tissues. The α5 chain is abundant in the perimysial regions and weakly expressed in endomysium. Both the α5 and α6 chains show partial co-localization with the α3 chain. Arrows point to selected regions that are exclusively positive for either the α5 or the α6 chain. The α4 chain is completely absent in skeletal muscle. J–L. Transversal section double labeled with the α5 chain antibody (J) and TRITC conjugated alpha-bungarotoxin (K) and merge image (L) showing the selective and strong expression of the α5 chain at the basement membrane of neuromuscular junctions. Nuclei were counterstained with bisbenzimidine (blue). Scale bar, 100 μm in A–I and 15 μm in J–L.

the wall of the follicle (Fig. 5B). In addition, the α4 chain is present in parts of the vascular medullar region. Interestingly, the staining for the α4 chain in ovary mostly co-localizes with that for the α3 chain (Fig. 5B,C). The α3 and α4 chains are also co-expressed in the outer connective tissue layer, the tunica albuginea. On the other hand, the α5 chain is only present in the stroma between follicles in the cortical region and co-localizes with the α3 chain (Fig. 5E,F). It is completely absent around the theca cells (Fig. 5E). Taken together, the expression patterns of the α4 and α5 chains in ovary are complementary (Fig. 5A–I) and that of the α6 chain coincides with α3, but is at the detection limit. In adult mouse testis, the α3 and α4 chains are widely expressed and completely co-localize. Both chains are expressed in the peripheral surrounding layer of testis, the tunica albuginea, which is a fibrous connective tissue primarily made up of collagens. In addition, both chains are significantly present in the thin loose fibrous connective tissue surrounding the seminiferous tubules (Fig. 45J–L). In contrast, the α5 chain is present in the tunica albuginea, where it clearly co-localizes with the α3 chain, but is absent in the connective tissue of seminiferous tubules (Fig. 5N,O). The α6 chain is only weakly present in the tunica albuginea but is clearly not expressed in the tissue surrounding seminiferous tubules (Fig. 5P–R).

2.7. Collagen VI expression in kidney Particularly selective and restricted expression patterns of the novel collagen VI chains were seen in kidney (Table 1). In sections of adult mouse kidney, the α3 chain is widely distributed in the cortical (Supplementary Fig. 3A,D,G) and medullar (not shown) regions. The capillary tufts/glomeruli and proximal convoluted tubules were labeled by the α3 chain antiserum. Interestingly, the α4 and α5 chains are highly restricted to the glomeruli where they are associated with basement membranes and do not completely co-localize with the α3 chain (Supplementary Fig. 3B,C,E,F). The expression of the α5 chain is stronger and more specific when compared to the α4 chain. The collagen VI α6 chain is completely absent in the cortical region and glomeruli (Supplementary Fig. 3H). The α3 chain is also strongly present in the kidney capsule. Here, the α5 chain partially co-localizes with the α3 chain (not shown). In addition, the α4 and α6 chains are weakly expressed in adult kidney capsule (not shown). 2.8. The novel chains are absent in cartilage The collagen VI α3 chain shows a very distinct and prominent expression in various layers of the growth plate of the femur. It is

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2.9. Western blot and immunoprecipitation analysis of collagen VI chains in tissue extracts Protein extracts from selected mouse tissues were analyzed by SDS-PAGE under reducing conditions followed by immunoblot with the specific antibodies to provide further characterization of the tissue forms of collagen VI and to confirm the results of the immunohistochemical analysis (Fig. 6A). Antibodies detecting the α1 were used as a control to show the restricted expression of the novel chains. Similar to the α1 chain, the α3 chain was detected in all tested tissues, but showed a variation in electrophoretic mobility from 260 kDa in testis to 220 kDa in quadriceps (Fig. 6A). The α4 chain migrates at 260 kDa and a strong signal was observed in intestine, ovary and testis, but was absent in quadriceps and heart. The α5 chain antibody detected a strong band at 260 kDa only in testis and weaker ones in intestine and ovary. After longer exposure bands appeared also in quadriceps and heart. Strong signals for the α6 chain at 260 kDa were identified in extracts from quadriceps, heart and ovary and weaker signals in testis. However, the α6 chain was absent in extracts of intestine. In addition, to study the composition of the collagen VI assembly forms in which the new chains occur, tissue extracts from ovary and testis were immunoprecipitated with a rabbit polyclonal antiserum against the pepsin-resistant form of murine collagen VI (Colombatti et al., 1992). Indeed, all the new chains were co-precipitated (Fig. 6B), indicating that these are part of the classical collagen VI microfibrils. The specificity of the immunoprecipitation was supported by the lack of clear signals in extracts obtained from Col6a1 knockout mice (Fig. 6B). 3. Discussion Fig. 3. Immunoelectron microscopy analysis of the collagen VI α6 chain in skeletal muscle. Ultrathin sections from adult mouse quadriceps were stained with gold-labeled antibodies against the collagen VI α6 chain (B, C, E and G) and the α1,2,3 chains (A, D and F). A. In the perimysium α1,2,3 chain gold-labeled antibodies (arrowheads) mainly bound to the microfibrillar network that encircles collagen fibers (cf). fb, fibroblast. B. Parallel running microfilaments (white arrows) labeled in a periodical pattern by α6conjugated colloidal gold particles (arrowheads) are visible in the perimysium C. α6 chain antibodies (arrowheads) were localized at the endomysium (en). D. α1,2,3 chain antibodies were detected at the reticular lamina associated with collagen fibrils (arrows), and at the interface of the lamina rara (r) with the lamina densa (d) (arrowheads). E. α6 chain antibodies (arrows) were absent at the interface of the lamina rara (r) and the lamina densa (d). F and G. α1,2,3 chains were detected at the basement membrane (bm) of capillaries (arrowheads) (F), while the α6 chain appears to be absent (G). The α6 chain (arrowheads) is associated with collagen fibers of the lamina rara of an adjacent myofiber (m). ec, endothelial cell. The inset in F shows an overview. Scale bars, 150 nm in B, 300 nm in A and C–G and 1 μm in inset of F).

weakly expressed in the resting zone and stronger in the proliferating and hypertrophic cartilage. However, none of the novel chains are present in the growth plate (Supplementary Fig. 5A–I). Interestingly, the α5 and α6 chains together with the α3 chain show a highly localized expression in a ligament (Supplementary Fig. 5D–I). In newborn mouse, the α3 chain is also broadly expressed in the growth plates of vertebrae and in the fibrocartilage of intervertebral disks (Supplementary Fig. 5J,M,P). Similar to the knee cartilage, the novel collagen VI chains are absent (Supplementary Fig. 5J–R). The new chains were also not detected in sternum, tongue, lung, liver, spleen, pancreas, and brain (not shown).

Three novel collagen VI chains were recently described, but only partially characterized (Fitzgerald et al., 2008; Gara et al., 2008). In the present study we analyzed their expression in mouse, using immunohistochemistry and immunoblots. As it is likely that the new chains replace the α3 chain, probably forming α1α2α4, α1α2α5 or α1α2α6 heterotrimers (Gara et al., 2008), we also generated antibodies specific for this chain to be able to distinguish between the different assembly forms. The novel chains are clearly less abundant than the broadly distributed α3 chain and show a very specific and narrow tissue distribution (Table 1). This could partially explain why these chains were not identified earlier, when the analysis was mainly performed at the protein level. Interestingly, at embryonic day 14.5 the expression is even sparser than after birth and restricted to bronchi, intestine and stomach (Fig. 1). In most cases, with the exception of embryonic stomach (Fig. 1N,O), the expression of the novel chains coincides with that of the α3 chain, which indicates that collagen VI molecules containing the new chains can be assembled into an α3 chain containing microfibrillar network. This indication is further supported by co-immunoprecipitation of the new chains by an antibody against the triple helical region of classical collagen VI (Fig. 6B). Immunostaining with the affinity purified antibodies of three different muscle types from both newborn and adult mice revealed that the expression of the novel chains in muscle does not change significantly after birth. However, the three new chains vary in their spatial expression pattern. The α4 chain is predominantly associated with basement membrane structures in the smooth muscle

Fig. 4. Immunohistochemical analysis of intestine and skin from adult mouse. Frozen sections of intestine (A–I) and skin (J–U) were incubated with affinity-purified antibodies against the collagen VI α3 (A, D, G and J, M, P, S, U), α4 (B and K), α5 (E and N), and α6 (H and Q) chains and CD31 (T and U) followed by AlexaFluor 488 (green) and 546 (red) labeled secondary antibodies. The merges of the stainings for the α4 (C and L), α5 (F and O) and α6 (I and R) chains and CD31 (U) with that for the α3 chain are shown for comparison. In the intestine (A–I), the α4 chain is expressed in the submucosal (sm), mucosal (m) and villi regions (v). s, sclerosa, ml, muscular layer. The α5 chain is strongly expressed in the mucosal layer and weakly present in villi. The α6 chain is not expressed in the intestine. In the skin (J–R), the α3 chain is widely distributed in the dermis (d) but absent in the epidermis (e). An arrowhead indicates the position of the dermal–epidermal basement membrane. The α4 chain shows a weak, patchy staining in the dermis (d). The weak staining of the epidermis (e) is not specific. The α5 chain shows restricted expression around blood vessels (arrows) and a patchy expression, probably at nerves or macrophages, in dermis. The α6 chain is absent in adult skin. At higher magnification (S–U) co-staining with CD31 antibodies clearly localizes the α5 chain in the vessel wall. Scale bar (A–R), 100 μm, (S–U), 50 μm.

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layers of intestine (Fig. 4B), but is absent in both skeletal and cardiac muscle of adult mice (Fig. 2B; Supplementary Fig. 2B). In contrast, the α6 chain is abundant and associated with both perimysial and epimysial basement membrane structures in skeletal and cardiac muscles, but does not show expression in smooth muscle (Fig. 2H; Fig. 4H; Supplementary Fig. 2H). Although the α5 chain is present in

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all three muscle types, the expression in skeletal and cardiac muscle is relatively weak compared to that of the α6 chain (Fig. 2E; Fig. 4E; Supplementary Fig. 2E), except for that the α5 chain is strongly expressed in the basement membrane at the neuromuscular junction (Fig. 2J–L). In newborn skeletal muscle, the α6 chain is restricted mainly to the endomysium. Interestingly, here the novel chains are

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Fig. 5. Immunohistochemical analysis of adult mouse ovary and testis. Frozen sections of ovary (A–I) and testis (J–R) were incubated with affinity-purified antibodies against the collagen VI α3 (A, D, G and J, M, P), α4 (B and K), α5 (E and N) and α6 (H and Q) chains followed by AlexaFluor 488 (green) and 546 (red) labeled secondary antibodies. The merges of the stainings for the α4 (C and L), α5 (F and O) and α6 (I and R) chains with that for the α3 chain are shown for comparison. In ovary (A–I) the α3 chain is widely distributed in tunica albuginea (ta), cortical stroma (c), medulla (m) and around theca cells (th). The α4 chain shows strong expression around theca cells and a weak expression in cortical stroma. The α5 chain antibodies stain predominantly in the cortical stroma and not around theca cells. The α6 chain is expressed at the detection limit in ovary. In testis (J–R), the α3 and α4 chains show strong expression and co-localize in tunica albuginea and in the loose connective tissue surrounding the seminiferous tubules (st). The α5 chain is restricted to tunica albuginea (ta) and co-localizes with the α3 chain. A very weak staining for the α6 chain is observed at the tunica albuginea. Scale bar, 100 μm.

only partially co-localized with the α3 chain indicating that some collagen VI microfibrils lack the α3 chain (not shown). Similarly, there is a partial co-localization of the α4 and α5 chains with the α3 chain in kidney and esophagus (Supplementary Fig. 3C,F and not shown). The α4 and α5 chains are present in adult mouse testis and this is the only tissue that exhibits an absolute co-localization with the α3 chain (Fig. 5J–O). The α5 chain is specifically associated with basement membranes surrounding blood vessels in the dermis (Fig. 4N,S,U). However, the α4 and α6 chains do not show specific staining at those

sites (Fig. 4K,Q). This differential and restricted distribution of the new collagen VI chains indicates that they may have tissue specific functions allowing a modulation of collagen VI properties. The existence of six different collagen VI chains is reminiscent to the basement membrane specific collagen IV, where also a “classical” widely distributed trimeric form exists (Khoshnoodi et al., 2008). However, the “classical” collagen VI is composed of three different chains α1, α2 and α3, whereas the “classical” collagen IV contains two α1 chains and one α2 chain. In contrast to collagen VI, the tissue

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Fig. 6. Immunoblot analysis and co-immunoprecipitation of collagen VI chains in extracts from selected tissues. A. Proteins were separated under reducing conditions and detected with polyclonal antibodies specific for the collagen VI α3, α4, α5 and α6 chains. The α1 chain was used for comparison. LE, longer exposure. B. Tissue extracts from ovary and testis of wild type (WT) and α1 chain collagen VI knockout (Col6a1−/−) mice were immunoprecipitated with a rabbit polyclonal antiserum against the pepsinresistant form of murine collagen VI. Immunoprecipitated proteins were separated under reducing conditions and detected with polyclonal antibodies specific for the collagen VI α3, α4, α5 and α6 chains.

specific forms of collagen IV lack the α1 and α2 chain and are formed by four different chains that assemble into α3α4α5 and α5α5α6 heterotrimers. Therefore collagen VI is the only collagen that forms different heterotrimers by the exchange of only one chain. Interestingly, the lack of collagen VI in Col6a1−/− mice leads to accelerated development of osteoarthritic joint degeneration (Alexopoulos et al., 2009) and it was shown that collagen VI is up-regulated in human osteoarthritis (Pullig et al., 1999). In contrast to the widely distributed collagen VI α3 chain, the novel chains are not expressed in mouse cartilage. Therefore, at least in mouse, the novel chains are not essential for the formation of a functional cartilage extracellular matrix. However, in contrast to our results from mouse, it was reported that the α6 chain is present in human articular cartilage and localized to the territorial matrix (Fitzgerald et al., 2008). Interestingly, the expression of the novel chains in skin also differs between man and mouse. Whereas the α5 chain in man is mainly expressed at the papillary dermis, with a more intense labeling just below the dermal–epidermal junction (Sabatelli et al., 2011), in mouse the α5 chain is associated with basement membranes surrounding blood vessels in the dermis similar to the distribution of the α6 chain in human skin. The differences in tissue distribution between man and mouse could be the result of the loss of a functional α4 chain gene in humans. Moreover, an aberrant epidermal localization has been reported for the human α5 chain (Söderhäll et al., 2007), which could not be confirmed in another study on human skin (Sabatelli et al., 2011) or in the present work on mouse. To our knowledge collagen VI has never been found in the epidermis by other authors. Generally, only a few collagens are located in the epidermis, e.g. collagen XXIII (Koch et al., 2006). The antibody that detected the α5 chain in epidermis was raised against a short peptide sequence. It is likely that the linear epitope of the peptide does not yield an antibody that detects conformational epitopes, as is needed for successful immunolocalization on tissue sections, and the epidermal staining by the peptide antibody could therefore be an artifact. The antibodies used in the present study were raised against large portions of N-terminal, non-triple helical domains of the collagen VI chains (Gara et al., 2008), expressed in a eukaryotic system to ensure correct folding and purified under native conditions. In addition, the antisera were affinity-purified against the protein that was used for immunization. The immunoblot analysis (Fig. 6A) of the novel chains in tissue extracts confirms the immunohistochemical findings. Whereas signals

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for the α1 and the α3 chains were detected in quadriceps, heart, intestine, ovary and testis, the α4 chain was absent in quadriceps and heart, and the α6 chain was absent in intestine. As was to be expected from the immunohistochemistry, signals for the α5 chain were weak in quadriceps and heart and could only be seen after overexposure. All bands for the new chains migrate around 260 kDa, which is close to the calculated masses of 248 kDa and 244 kDa for the α4 chain and the α6 chain, respectively, but not to that of the calculated mass of 287 kDa for the α5 chain. As we were not able to identify a corresponding alternatively spliced mRNA (not shown), proteolytic processing could be the reason for the shorter products, and indeed, in the overexposed blot a band migrating above 260 kDa was detected. Interestingly, although the expression level was generally weak in quadriceps the strongest band clearly migrates below 260 kDa, which could point to a specific form of the α5 chain in skeletal muscle. Taken together, the results show that the novel chains are closely associated with basement membrane structures in several tissues, pointing to a potential role in anchoring epithelia, nerves and blood vessels to the extracellular matrix. Further characterization of the novel collagen VI chains will be aimed at understanding their roles in development, tissue homeostasis and the pathogenesis of inherited disease. 4. Experimental procedures 4.1. Recombinant expression and purification of N-terminal collagen VI α3 chain VWA domains The cDNA construct coding for the N4–N10 domains was generated by RT-PCR on total RNA from brain and cloned with 5′terminal NheI and 3′-terminal XhoI restriction sites using the primers a3m1(f) 5′-AAA GCT AGC ACA ACA GCA TGG AGA TGT CAA AA-3′ and a3m2(r) 5′-TAT CTC GAG CTG TGA GGT TAG AGT GGT GAT G-3′. The amplified PCR product was inserted into a modified pCEP-Pu vector containing an N-terminal BM-40 signal peptide and a C-terminal tandem strepII-tag downstream of the restriction sites (Maertens et al., 2007). The recombinant plasmids were introduced into HEK293-EBNA cells (Invitrogen) using FuGENE 6 transfection reagents (Roche). The cells were selected with puromycin (1 μg/ml) and the recombinant protein was purified directly from serum-containing cell culture medium. After filtration and centrifugation (1 h, 10,000 × g), the cell culture supernatants were applied to a Streptactin column (1.5 ml, IBA GmbH) and eluted with 2.5 mM desthiobiotin, 10 mM Tris–HCl, pH 8.0. 4.2. Preparation of specific antibodies The purified recombinant collagen VI α3 (N4–N10) fragment, was used to immunize a rabbit and a guinea pig. The antisera obtained were purified by affinity chromatography on a column with antigen coupled to CNBr-activated Sepharose (GE Healthcare). The specific antibodies were eluted with 0.1 M glycine, pH 2.5, and the eluate was neutralized with 1 M Tris–HCl, pH 8.8. The preparation of antibodies against the mouse collagen VI α4, α5 and α6 chains was described earlier (Gara et al., 2008). 4.3. Immunohistochemistry Immunohistochemistry was performed on frozen embedded sections from 14.5-day-old mouse embryos and newborn and adult mice. The frozen sections were left at room temperature for 1 h, preincubated in phosphate-buffered saline (PBS) for 5 min and then fixed with 2% paraformaldehyde in PBS for 10 min and washed three times in PBS for 5 min each. The sections were incubated with freshly prepared hyaluronidase (50 U/ml) for 30 min at 37 °C, washed twice with Tris-buffered saline (TBS) for 5 min each and blocked for 1 h

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with 5% BSA/0.1% Triton-X100 in TBS at room temperature. The primary antibodies were diluted in 1% BSA/TBS and applied for 1 h at 37 °C and washed three times for 5 min each with 0.1% Triton/TBS. For co-staining with CD31 a purified rat anti-mouse CD31 monoclonal antibody was used (BD Pharmingen). The sections were then incubated for 1 h in the dark with AlexaFluor 546-conjugated goat anti-rabbit IgG (Molecular Probes), AlexaFluor 488-conjugated goat anti-guinea pig IgG (Molecular Probes), or AlexaFluor 546-conjugated goat anti-rat IgG (Molecular Probes). For co-staining of the neuromuscular junction after immunolabeling with the α5 chain antibody, skeletal muscle sections were incubated with TRITC conjugated alphabungarotoxin (Molecular Probes) diluted 1/1000 in PBS for 1 h at room temperature. After washing three times, the slides were incubated for 5 min in 1 μg/ml bisbenzimidine/ethanol. The sections were washed three times and then mounted with Dako fluorescent mounting medium. Pictures were taken by conventional immunofluorescence microscopy. 4.4. Preparation of protein extracts for immunoblotting Adult mouse tissues frozen in liquid nitrogen were pulverized by pestle and mortar and thawed and lysed in 10 mM MgCl2, 0.5 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 2% SDS, 1% Triton X-100 and Complete protease inhibitors (Roche). Proteins were solubilized by heating at 70 °C for 10 min. Samples were clarified by centrifugation at 4 °C, reduced with 5% β-mercaptoethanol and subjected to SDSPAGE on 3–8% (w/v) gradient polyacrylamide gels. Proteins were electrophoretically transferred to Immobilon-P membranes (Millipore). The collagen VI α1 chain was detected using a polyclonal antibody against the human chain (H-200, Santa Cruz Biotechnology) and the α3, α4, α5, and α6 chains with the affinity-purified antibodies described above. Secondary antibodies conjugated with horseradish peroxidase were used, and bands were detected by chemiluminescence (SuperSignal West Pico, Pierce). 4.5. Preparation of protein extracts for immunoprecipitation Mouse tissues frozen in liquid nitrogen were pulverized by pestle and mortar and thawed and lysed in 20 mM Tris–HCl pH 7.6, 100 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Nonidet P 40, 1% sodium deoxycholate, and Complete protease inhibitors (Roche). Proteins were solubilized by heating at 70 °C for 10 min. and samples were clarified by centrifugation at 4 °C. The samples were first precleared by incubation with 100 μl protein A-Sepharose (Santa Cruz Biotechnologies), 2 mg/ml bovine serum albumin and 4 μl pre-immune serum over night at 4 °C. After centrifugation for 10 min at 500 ×g, 2 μl of the mouse collagen VI specific AS-72 antibody (a gift from A. Colombatti) (Colombatti et al. 1992), and 50 μl of protein A-Sepharose were added to the supernatant, and the incubation continued over night at 4 °C. The samples were centrifuged at 500 × g and the precipitates washed three times with PBS containing 10% CHAPS (Sigma) prior to dissolving in Laemmli sample buffer in the presence of 5% (v/v) 2β-mercaptoethanol. Immunoprecipitated proteins were further reduced with 10% DTT and subjected to SDS-PAGE on 3–8% (w/v) gradient polyacrylamide gels. Proteins were electrophoretically transferred to Immobilon-P membranes (Millipore). The collagen VI α3, α4, α5, and α6 chains were detected with the affinity-purified antibodies described above. Secondary antibodies conjugated with horseradish peroxidase were used, and bands were detected by chemiluminescence (SuperSignal West Pico, Pierce). 4.6. Immunoelectron microscopy En bloc immunolabeling of skeletal muscle was performed as previously reported (Sakai et al., 1986) with some modifications. Freshly obtained quadriceps were minced in PBS, pH 7.4, and

incubated with the α1,2,3 chain (70R-CR009x, Fitzgerald Industries International) or the α6 chain antibody (diluted 1/10 in culture medium DMEM with 20% fetal calf serum) over night at 4 °C. After several washings with DMEM, the samples were incubated with 5 nm colloidal gold conjugated anti-rabbit IgG antibodies (Sigma) for 1 h at room temperature. After extensive washing with PBS, the samples were fixed with 2.5% glutaraldehyde in phosphate buffer and postfixed with 1% osmium tetroxide in Veronal buffer, dehydrated and embedded in Epon812. Before conventional staining with uranyl acetate and lead citrate, ultrathin sections were treated with a silver enhancement kit (BBI International) to increase the size of the colloidal gold particles, and observed with a transmission electron microscope Philips EM400 operated at 100 kV. Acknowledgments This study was supported by the Deutsche Forschungsgemeinschaft (WA1338/2-6 and SFB 829) to RW and MP and by the Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale (PRIN 2008 grant no. 2008PB5S89) to PS, SS and PB. SKG was a member of the International Graduate School in Genetics and Functional Genomics at the University of Cologne. Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.matbio.2011.03.006. References Alexopoulos, L.G., Youn, I., Bonaldo, P., Guilak, F., 2009. Developmental and osteoarthritic changes in Col6a1-knockout mice: biomechanics of type VI collagen in the cartilage pericellular matrix. Arthritis Rheum. 60, 771–779. Castro-Giner, F., Bustamante, M., Ramon, G.J., Kogevinas, M., Jarvis, D., Heinrich, J., Anto, J.M., Wjst, M., Estivill, X., de, C.R., 2009. A pooling-based genome-wide analysis identifies new potential candidate genes for atopy in the European Community Respiratory Health Survey (ECRHS). BMC Med. Genet. 10, 128. Chu, M.L., Conway, D., Pan, T.C., Baldwin, C., Mann, K., Deutzmann, R., Timpl, R., 1988. Amino acid sequence of the triple-helical domain of human collagen type VI. J. Biol. Chem. 263, 18601–18606. Chung, E., Rhodes, K., Miller, E.J., 1976. Isolation of three collagenous components of probable basement membrane origin from several tissues. Biochem. Biophys. Res. Commun. 71, 1167–1174. Colombatti, A., Bonaldo, P., Bucciotti, F., 1992. Stable expression of chicken type-VI collagen alpha 1, alpha 2 and alpha 3 cDNAs in murine NIH/3T3 cells. Eur. J. Biochem. 209, 785–792. Fitzgerald, J., Rich, C., Zhou, F.H., Hansen, U., 2008. Three novel collagen VI chains, alpha 4(VI), alpha 5(VI) and alpha 6(VI). J. Biol. Chem. 283, 20170–20180. Furthmayr, H., Wiedemann, H., Timpl, R., Odermatt, E., Engel, J., 1983. Electronmicroscopical approach to a structural model of intima collagen. Biochem. J. 211, 303–311. Furuto, D.K., Miller, E.J., 1980. Isolation of a unique collagenous fraction from limited pepsin digests of human placental tissue. Characterization of one of the constituent polypeptide chains. J. Biol. Chem. 255, 290–295. Furuto, D.K., Miller, E.J., 1981. Characterization of a unique collagenous fraction from limited pepsin digests of human placental tissue: molecular organization of the native aggregate. Biochemistry 20, 1635–1640. Gara, S.K., Grumati, P., Urciuolo, A., Bonaldo, P., Kobbe, B., Koch, M., Paulsson, M., Wagener, R., 2008. Three novel collagen VI chains with high homology to the alpha3 chain. J. Biol. Chem. 283, 10658–10670. Grumati, P., Coletto, L., Sabatelli, P., Cescon, M., Angelin, A., Bertaggia, E., Blaauw, B., Urciuolo, A., Tiepolo, T., Merlini, L., Maraldi, N.M., Bernardi, P., Sandri, M., Bonaldo, P., 2010. Autophagy is defective in collagen VI muscular dystrophies, and its reactivation rescues myofiber degeneration. Nat. Med. 16, 1313–1320. Harazin, M., Parwez, Q., Petrasch-Parwez, E., Epplen, J.T., Arinir, U., Hoffjan, S., Stemmler, S., 2010. Variation in the COL29A1 gene in German patients with atopic dermatitis, asthma and chronic obstructive pulmonary disease. J. Dermatol. 37, 740–742. Jander, R., Rauterberg, J., Voss, B., von Bassewitz, D.B., 1981. A cysteine-rich collagenous protein from bovine placenta. Isolation of its constituent polypeptide chains and some properties of the non-denatured protein. Eur. J. Biochem. 114, 17–25. Jander, R., Rauterberg, J., Glanville, R.W., 1983. Further characterization of the three polypeptide chains of bovine and human short-chain collagen (intima collagen). Eur. J. Biochem. 133, 39–46. Khoshnoodi, J., Pedchenko, V., Hudson, B.G., 2008. Mammalian collagen IV. Microsc. Res. Tech. 71, 357–370.

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