Discrete integration of collagen XVI into tissue-specific collagen fibrils or beaded microfibrils

Matrix Biology 22 (2003) 131–143

Discrete integration of collagen XVI into tissue-specific collagen fibrils or beaded microfibrils Anja Kassnera,1, Uwe Hansena,1, Nicolai Miosgeb, Dieter P. Reinhardtc, Thomas Aignerd, a, ¨ Leena Bruckner-Tudermane, Peter Brucknera, Susanne Grassel * a

¨ Physiologische Chemie und Pathobiochemie, Universitatsklinikum ¨ ¨ ¨ Institut fur Munster, 48129 Munster, Germany b ¨ Gottingen, ¨ ¨ Anatomie und Histologie, 37075 Gottingen, ¨ Universitat Institut fur Germany c ¨ Lubeck, ¨ ¨ Medizinische Molekularbiologie, 23538 Lubeck, ¨ Medizinische Universitat Institut fur Germany d ¨ Erlangen, Institut fur ¨ Pathologie, 91054 Erlangen, Germany Universitat e ¨ ¨ ¨ Hautklinik, Universitatsklinikum Munster, 48129 Munster, Germany Received 10 October 2002; received in revised form 2 January 2003; accepted 6 January 2003

Abstract The structural and functional diversity of extracellular matrices is determined, not only by individual macromolecules, but even more decisively, by the alloyed aggregates they form. Although quantitatively major matrix molecules can occur ubiquitously, their organization varies from one tissue to another due to their amalgamation with specific sets of minor components. Here, we show that the fibril-associated collagen with interrupted triple helices collagen XVI is unique in that, depending on the tissue context, it can be incorporated into distinct suprastructural aggregates. In papillary dermis, the protein unexpectedly does not occur in banded collagen fibrils, but rather, is a component of specialized fibrillin-1-containing microfibrils. In territorial cartilage matrix, however, collagen XVI is not a component of aggregates containing fibrillin-1. Instead, the protein resides in a discrete population of thin, weakly banded collagen fibrils also containing collagens II and XI. Collagen IX also occurs in this population of fibrils, but at longitudinal locations discrete from those of collagen XVI. This suprastructural versatility of a collagen is without precedent and highlights pivotal differences in the tissue-specific organization of matrix aggregate structures. 䊚 2003 Elsevier Science B.V.yInternational Society of Matrix Biology. All rights reserved. Keywords: Cartilage fibrils; Microfibrils; Collagen IX; Fibrillin-1; Dermo-epidermal junction

1. Introduction Interactions of cells with the extracellular matrix (ECM) contribute to the specification of cellular responses essential for development, growth, maintenance and repair of tissues and organs. The structural determinants of the extracellular ligands reside not only in individual matrix macromolecules, but also in the surface of aggregates they form. Supramolecular organizations vary from one tissue to another or within regions of a given tissue. For example, the quantitative major structural components of banded collagen fibrils are collagens with a single triple helical domain covering a ¨ Physiologische Chemie und *Corresponding author: Institut fur ¨ Pathobiochemie, Waldeyerstr. 15, 48149 Munster, Germany. Tel.: q 49-251-8355574; fax: q49-251-8355596. ¨ E-mail address: [email protected] (S. Grassel). 1 Both authors contributed equally to this work.

length of 300 nm. However, these collagens are amalgamated with minor constituents in discrete tissuespecific arrays. The structural particularities of these fibrils manifest, among others, in banding periodicities D varying between 64 and 67 nm from one tissue to another and in lateral spacings of collagen molecules between 1.5 and 1.7 nm implying distinct molecular organizations (Brodsky and Eikenberry, 1985; Eikenberry and Bruckner, 1999; van der Rest and Bruckner, 1993). Similarly, beaded microfibrils contain fibrillins as major components, but also have variable molecular organizations (Baldock et al., 2001; Sherratt et al., 2001). Dermal matrix contains lose bundles of fibrils rich in collagen I. Even as a pure protein, collagen I can selfassemble into cross-banded fibrils and, for this reason, has been designated as a fibril-forming collagen. However, the protein in skin is a constituent of complex,

0945-053X/03/$30.00 䊚 2003 Elsevier Science B.V.yInternational Society of Matrix Biology. All rights reserved. doi:10.1016/S0945-053X(03)00008-8

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heterotypic fibrils containing collagens III and V, as well as XII and XIV in tissue-specific ratios. In contrast to collagens I, III and V, collagens XII and XIV cannot form aggregates by themselves and have relatively short triple helical domains. Although some of these domains are integrated into the fibrils in situ, thereby orchestrating andyor stabilizing the fibrillar organization of the major collagens, collagens XII and XIV have been designated as fibril-associated collagens with interrupted triple helices (FACIT) (for review, see Birk and Mayne (1997), Shaw and Olsen (1991) and van der Rest and Bruckner (1993)). In addition to collagens, dermal fibrils also incorporate non-collagenous molecules, notably decorin, a small, leucine-rich repeat—proteoglycan (Iozzo, 1998). The dermatan sulfate chains of decorin impart a high negative charge density to the fibril surface and are thought to serve as spacers between individual fibrils within the bundles and so to prevent their lateral fusion (Scott, 1992, 1995). Dermal connective tissue is permeated by elastic fibres that can be separated from collagen-containing fibrils, e.g. by collagenase treatment. Elastic fibres confer to this tissue its long range-elasticity and contain elastin as the major molecular constituent. They are stabilized by beaded microfibrils with a periodicity of 50–55 nm that also act as templates for tropoelastin deposition during elastic fibrillogenesis (Handford et al., 2000). Hence, mature elastic fibres in most tissues are composites of elastin and microfibrils (Mecham and Heuser, 1990). Microfibrils, too, are molecular alloys, fibrillins-1 and -2 being the quantitatively major molecular constituents. Fibrillin-1 contributes to both, the beads and the filamentous strands of the network (Reinhardt et al., 1996a). Quick-freeze deep-etch images have revealed microfibrils with a mean string diameter of 10–12 nm (Mecham and Heuser, 1991) whereas 30–40 nm were measured for the bead diameters by atomic force microscopy (Hanssen et al., 1998). Depending on tissue or developmental stage, microfibrils comprise several other matrix molecules including microfibrilassociated glycoproteins (MAGPs 1 and 2) (Gibson et al., 1998; Henderson et al., 1996), several latent TGFb binding proteins (LTBPs) (Raghunath et al., 1998; Taipale et al., 1996), fibulin-2 (Reinhardt et al., 1996b) and versican (Isogai et al., 2002). In addition, a panel of other components have been tentatively assigned to the supramolecular structure of microfibrils. However, regardless of tissue occurrence and developmental stage, microfibrillar alloys have not been reported to contain collagens. In hyaline cartilage, the ECM occupies the largest volume fraction and consists of a fibrillar network entrapping a highly hydrated proteoglycan matrix. Cartilage fibrils contain several macromolecules that are typical, if not specific for this tissue. The major cartilage collagen is collagen II, which, in spite of its high homology to collagen I, does not form fibrils by itself

in vitro, but very inefficiently assembles into tactoids, i.e., D-periodically banded aggregates of limited length and lacking the stringent width-control typical for cartilage fibrils (Blaschke et al., 2000; Lee and Piez, 1983). In situ, collagen II is a component of heterotypic fibrils also containing collagen XI, a protein strongly enhancing fibril formation. Notably, fibrillogenesis of collagen IIy XI-alloys in vitro is tightly regulated in that, fibrils with a width of 20 nm are formed exclusively (Blaschke et al., 2000). Depending on their precise location in the tissue, cartilage fibrils also harbour several other macromolecules including collagen IX, or proteoglycans such as decorin and biglycan (for review, see Eikenberry and Bruckner, 1999). Collagens XII and XIV also are cartilage components and are fibril-associated (Gregory et al., 2001; Watt et al., 1992). Although they represent a sizable fraction of matrix macromolecules their precise function in cartilage still remains to be defined. Collagen XVI is a structural analogue of the FACIT— collagens IX, XII, XIV and XIX (Prockop and Kivirikko, 1995; van der Rest and Bruckner, 1993) and is another component of several connective tissues. During early mouse development, collagen XVI occurs in many tissues and is co-distributed with the major fibrillar collagens. In particular, collagen XVI is strongly expressed in differentiating chondrocytes and skin (Lai and Chu, 1996). In skin, collagen XVI preferentially occurs in narrow zones near basement membranes at the dermo-epidermal junction (DEJ) or of blood vessels ¨ (Grassel et al., 1999). This feature is preserved by dermal fibroblasts in culture. Cells explanted from the superficial layers of the dermis produce more collagen XVI than fibroblasts derived from deeper dermal regions (Akagi et al., 1999). However, for no tissue the supramolecular organization of collagen XVI is known. Here, we show that the protein can be incorporated tissuespecifically into structurally and functionally discrete matrix aggregates in skin and cartilage. This suprastructural versatility is without precedent for collagens. 2. Results 2.1. Analysis of collagen XVI expression in skin and cartilage Collagen XVI-gene expression was analyzed by RNAextraction and RT-PCR in chondrocytes freshly isolated from articular cartilage of patients undergoing hip or knee joint replacement surgery. The size and intensity of the PCR product was compared with that obtained from dermal fibroblasts. A single band of approximately 340 bp resulted from amplification with a collagen XVI specific primer-pair (Fig. 1A, lane 2). This amplification product was similar to that obtained from primary human dermal fibroblasts (Fig. 1A, lane 3). GAPDH-expression

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juvenile ribs or adult joints, as well as from human dermis were analyzed by immuno-blotting. Single bands migrating slightly above the 209 kDa-standard reacted with a collagen XVI-specific antibody raised against the NC 11 domain (Eq) (Fig. 1B). This molecular mass was consistent with that reported previously for the a ¨ 1(XVI)-chains from fibroblast cultures (Grassel et al., 1996). The collagen XVI-specific band from knee joint extract (lane 1) exhibited a slightly larger molecular mass than that of dermis (lane 2). This variability may be explained by tissue-specific differences in glycosylation patterns or proteolytic processing. 2.2. Immunohistochemical analysis of human dermis and human costal cartilage with confocal laser scanning microscopy

Fig. 1. Detection of collagen XVI expression in cartilage. (A) RTPCR performed with total RNA extracted from freshly isolated chondrocytes from human adult knee joint cartilage (lanes 2 and 4) and with total RNA extracted from primary dermal human fibroblasts (lanes 3 and 5). PCR-reactions performed with primer pairs specific for collagen XVI result in one band at the expected size of 340 base pairs for both cartilage and fibroblast RNA (lanes 2 and 3). Control reactions were performed with primer pairs specific for GAPDH (lanes 4 and 5). Lane 1: 100 base pair (bp) DNA-ladder. (B) Western blot analysis of protein precipitates from human adult knee joint cartilage (lane 1) and human dermis (lane 2). One-fifth of the proteins precipitated from cartilage were loaded on lane 1 and total proteins precipitated from dermis were loaded on lane 2. Detection was performed with an antibody raised against the NC11 domain of collagen XVI (Eq) which recognizes one single protein band between 210 and 220 kDa.

Due to antigen masking in paraffin embedded sections cryosections were exclusively used for the following light microscopic studies. Staining of 4-mm cryosections from normal human skin with the collagen XVI specific antibody Eq revealed a strong signal along the DEJ zone and around blood vessels (Fig. 2). Only occasionally, the remaining interstitial matrix of the deeper dermal layers was collagen XVI-positive. Fibrillin-1, as detected by the monoclonal antibody Mab 69, had a more widespread distribution, extending also into the deeper layers of the dermis (Fig. 2A and B). Interestingly, we found a strong co-localization of fibrillin-1 and collagen XVI in the DEJ zone (Fig. 2A and B). In the remaining dermal matrix, the proteins occur discretely except for the basement membrane zones around blood vessels (Fig. 2A). Co-staining with antibodies to decorin and collagen XVI demonstrated distinct distribution patterns (Fig. 2C). Decorin, a typical component of collagen I-containing fibrils, is found throughout the dermis including the area of the DEJ where it showed only an occasional co-localization with collagen XVI. Staining of 4-mm cryosections from fetal costal cartilage with the anti-collagen XVI-antibody Eq revealed a strong fluorescence signal in the pericellular and territorial region of the chondrocytes. The interterritorial matrix remains unstained and, hence, appears to lack collagen XVI (Fig. 3A and B). The fibrillin-1 antibodies Mab 69 (Fig. 3C) and Mab 201 (not shown) exhibited a territorial and weak interterritorial staining pattern. Double-immunofluorescence microscopy indicated no significant co-localization of these antigens, although both, collagen XVI and fibrillin-1 occurred within the territorial matrix (Fig. 3A). 2.3. Supramolecular distribution and localization of collagen XVI in human skin and articular cartilage

was monitored as a control for RNA quality and PCR performance (Fig. 1A, lanes 4 and 5). In order to demonstrate collagen XVI-expression at the protein level, extracts from cartilage of human

In order to assess the organization of collagen XVI in the extracellular matrices of skin and cartilage ultrathin tissue sections were prepared and were stained by

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Fig. 2. Localization of collagen XVI in skin by confocal laser scanning microscopy. Cryosections of adult human skin were double labeled with antibodies against fibrillin-1 (Mab 69) and collagen XVI (Eq) (A and B) and against decorin and collagen XVI (Eq) (C). Collagen XVI is represented by red fluorescence, fibrillin-1 and decorin, are represented by green fluorescence. Overlapping areas of individual proteins are indicated by yellow fluorescence. Collagen XVI clearly co-localizes with fibrillin-1 in the DEJ zone and around blood vessels in deeper dermal layers, but not in other areas of the dermis (A, B). Decorin and collagen XVI show only minor co-localization in the DEJ zone, but generally appear in an alternating distribution pattern (C). bars100 mm (A), barss20 mm (B, C). de, dermis; ep, epidermis; asterisks: indicating the DEJ; arrows: indicating small capillaries.

immunogold-labeling (Figs. 4 and 5). Consistent with our observations by immunofluorescence in skin, abundant collagen XVI-clusters were localized to thin, dermal microfibrils in close vicinity to the dermo-epidermal

basement membrane, but not within deeper epidermal layers (Fig. 4A). In some areas, the occurrence of collagen XVI reached the zone of the lamina densa, but not beyond into the lamina lucida. Double immunogold-

Fig. 3. Localization of collagen XVI in cartilage by confocal laser scanning microscopy. Cryosections of fetal human costal cartilage were double labeled with antibodies against fibrillin-1 (Mab 69) and collagen type XVI (Eq) (A) and were individually labeled with each antibody either with Eq (B) or Mab 69 (C). Both, collagen XVI and fibrillin-1 reveal similar distribution patterns which for collagen XVI are restricted to the territorial matrix of individual chondrocytes, whereas fibrillin-1 is also found in the interterritorium. However, both proteins do not co-localize (A). barss30 mm; territorial matrix: encircled regions closely surrounding individual chondrocytes; interterritorial matrix: regions in between individual chondrocytes highlighted with asterisks.

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Fig. 4. Immunogold electron microscopy of ultrathin sections of adult human skin. K, keratinocyte, asterisks, dermo-epidermal basement membrane. (A) Collagen XVI is found in the basement membrane and in the fibrillar material underneath, bars0.25 mm. (B) Double labeling of collagen XVI (large gold particles, closed arrows) and fibrillin-1 (small gold particles, open arrows) are found co-localized in the area of the DEJ zone, bars0.16 mm. (C) Collagen XVI is associated to electron dense amorphous material (closed arrows), but not to the large D-periodically banded collagen containing fibrils (open arrows) of the papillary dermis, bars0.25 mm.

labeling with the antibody Mab 69 demonstrated fibrillin-1 location in the same area and corroborated an association of collagen XVI with selected parts of the microfibrillar apparatus of the skin, whereas other

suprastructurally similar microfibrils showed no collagen XVI-labeling (Fig. 4B). In addition, we have found clusters of collagen XVI antibodies associated to electron-optically amorphous suprastructures, partially inter-

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Immunogold-labeling of ultrathin sections of articular cartilage confirmed the occurrence and the distribution of collagen XVI in this tissue. The protein was found in territorial matrix regions on thin, weakly banded collagen-fibrils which were similar in appearance to heterotypic fibrils comprising collagens II, IX and XI, but not decorin (Hagg et al., 1998) (Fig. 5A). Therefore, collagen IX-containing fibrils may or may not incorporate collagen XVI as well. To distinguish between these possibilities, we performed double-immunogold-labeling with type-specific antibodies. A mutually exclusive suprastructural association of collagens IX and XVI was observed (Fig. 5C) indicating that territorial cartilage matrix encompassed at least two subpopulations of thin collagen fibrils with discrete molecular compositions. Further, the collagen–fibrillar networks in cartilage matrix were intermingled with broad banded fibres. These were similar to densely packed and laterally crosslinked fibrils reported to be unique for cartilage and to contain fibrillin-1, but not collagens II, VI, IX, XI or XII (Keene et al., 1997). None of these fibrillin1 positive fibres were labeled with collagen XVIantibodies (Fig. 5B). Taken together, these observations demonstrated that, unlike in skin, collagen XVI was a component of collagen fibrils in cartilage. Conversely, the protein was not a constituent in cartilage of fibrillin-1-containing suprastructures as was observed for some specialized dermal microfibrils. 2.4. Localization of collagen XVI in suprastructural fragments isolated from the extracellular matrices of human skin and cartilage

Fig. 5. Immunogold electron microscopy of ultrathin sections of adult articular cartilage from the radial zone. (A) Collagen XVI is found on D-periodically banded cartilage fibrils (closed arrows), bars0.25 mm. (B) Double labeling of collagen XVI (large gold particles, black arrows) and fibrillin-1 (small gold particles, open arrows) are found not to be co-localized, both are localized to different fibrillar systems, bars0.18 mm. (C) Double labeling of collagen XVI (large gold particles, closed arrows) and collagen IX (small gold particles, open arrows) are found not to be co-localized, both are associated to different D-periodically banded fibrillar fragments, bars0.18 mm.

secting the lamina densa, indicating that other not yet identified macromolecular composites exist which contain collagen XVI (Fig. 4B). Notably, however, the large banded collagen I- and decorin containing fibrils in the DEJ zone were collagen XVI-negative (Fig. 4C).

Immunohistological co-localization by light or electron microscopy does not necessarily imply the existence of physical interactions between macromolecules. For this reason, we conducted experiments with isolated fragments derived from authentic matrix suprastructures. Genuine components of matrix aggregates are retained during the isolation procedures and, hence, are amenable to identification by immunoelectron microscopy. Loosely associated constituents are eliminated in this process. Epidermal layers were removed from adult human skin and fibrillar extracts were prepared from the upper dermal regions (200–300 mm), including parts of the basement membrane and the papillary dermis. After negative staining with uranyl acetate, fibrillar suprastructures including large D-periodically banded fibrils of 60–100 nm in diameter were distinguishable by transmission electron microscopy from non-fibrillar components of the ECM (Fig. 6). Indirect immunogoldlabeling (18-nm gold-particles) was performed with collagen XVI-specific antibodies. Collagen XVI was associated with microfibrils (diameter -20 nm) appearing as beaded strings and without a D-periodic banding

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Fig. 6. Ultrastructural localization of collagen XVI in fibrillar extracts from skin by immunogold electron microscopy. (A) Thin, fibrillar structures are labeled with collagen XVI (Eq, l8-nm gold particles), large D-periodically banded collagen fibrils are collagen type XVInegative, bars0.18 mm. (B) Double labeling of collagen XVI (Eq, 18-nm gold particles, light arrow) and fibrillin-1 (Mab 69 or Mab 201, 12-nm gold particles) demonstrate co-localization on ‘bead on the string’ microfibrils with collagen XVI bound at one distinct bead of the microfibrils, bars0.12 mm. (C) The large D-periodically banded collagen fibril lacks collagen XVI labeling (18-nm gold particles, light arrow), instead the fibrillin-1 labeled microfibril (12-nm gold particles) is collagen XVI-positive, bars0.12 mm.

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pattern whereas the large, banded collagen fibrils were not labeled (Fig. 6A) corroborating the distribution pattern on the ultrathin tissue sections. Double-immunogold labeling of fibrillar extracts with anti-fibrillin-1 antibodies (12-nm gold-particles) and anti-collagen XVI antibodies (18-nm gold-particles) identified the major component of the microfibrils as fibrillin-1. Thus, collagen XVI was identified as a component of the microfibrillar apparatus in skin (Fig. 6B and C). Notably, collagen XVI did not occur uniformly along the fibrillin1-containing microfibrils but forms clusters preferentially at a distinct site at the globular beads. However, a majority of fibrillin-1-containing microfibrils, particularly those associated with a dense elastin core, was not labeled with antibodies to collagen XVI, indicating that only a relatively small subpopulation of dermal microfibrils contained collagen XVI. The suprastructural distribution of collagen XVI in cartilage was also investigated in supramolecular fragments from juvenile costal cartilage (Fig. 7). As in dermal extracts, non-fibrillar matrix-aggregates were clearly distinguishable from the more abundant fragments of D-periodically banded fibrils. The vast majority of the fibril fragments had a diameter of 15–30 nm and are known to contain collagens II, IX and XI (Hagg et al., 1998). They represented a population characteristic of immature cartilage since adult cartilage is rich in fibrils with larger diameters lacking collagen IX, but containing decorin. In striking contrast to its occurrence in skin, collagen XVI was localized mainly to the surface of the D-periodically banded cartilage collagen fibrils. Immunogold clusters were seen on the fibrils with a spacing of approximately 100–200 nm (Fig. 7). However, only a fraction of the D-periodically banded thin fibril fragments were labeled with antibodies to collagen XVI (Fig. 7A and B). Double immunolabeling revealed that labeling with antibodies to collagens IX and XVI occurred at distinct sites in a specialized fraction of fibril fragments (Fig. 7D and E) whereas a significant population of fragments were positive in a mutually exclusive manner for one of the proteins only (data not shown). In addition to the predominating fibril fragments, we also identified fibrillin-1-containing fibres (Fig. 7C). Their suprastructure appeared to differ from that of dermal microfibrils in that they lack the typical ‘beads on the string’ appearance. Interestingly, none of the cartilage fibrillin-1-containing fibres was associated with collagen XVI, indicating that their supramolecular composition differed profoundly from that of dermal microfibrils. 3. Discussion It is now well recognized that the multitude in extracellular matrices of fibrillar organizations and networks results from the fact that most, if not all, matrix

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Fig. 7. Ultrastructural localization of collagen XVI in fibrillar extracts from cartilage by immunogold electron microscopy. (A) A special subpopulation of thin D-periodically banded cartilage fibrils were labeled with collagen XVI (Eq, 18-nm gold particles), other fibril populations lack collagen XVI association, bars0.24 mm. (B) Larger magnification demonstrates association of collagen XVI with a linear spacing of approximately 100–200 nm along a particular cartilage fibril, bars0.09 mm. (C) Double immunogold-labeling with fibrillin-1 (Mab 69 or Mab 201, 12-nm gold particles and light arrows) and collagen XVI (Eq, 18-nm gold particles and closed arrows). Fibrillin-1 and collagen XVI stain different fibrillar systems, both proteins are not co-localized, bars0.12 mm. (D, E) Double immunogold-labeling with collagen IX (Mab D1-9 and Mab B3-1, 12-nm gold particles, light arrows) and collagen XVI (Eq, 18-nm gold particles, closed arrows). Collagen IX and XVI can be found associated with the same fibril, but always regionally separated, or on distinct fibrils, bars0.18 mm.

suprastructures are composed of more than one type of macromolecules. Thus, malleability of matrix structures is introduced by the formation of alloy-like aggregates in which major and minor components are functionally equivalent in generating structures that cannot be formed by the individual components alone or by mixtures other than the appropriate ones. However, so far it has not been known that one type of collagen can be a component of two structurally and functionally different categories of suprastructures. Here, we identify collagen XVI as the first molecule which plays this dual role in contributing to the structure of both, small D-periodic cartilage fibrils and of microfibrils in the DEJ zone.

In immature cartilages, fibrils with a characteristically uniform diameter of 20 nm and a weak banding pattern are alloys of collagen II and XI at a molar ratio of 8:1. Other components, such as collagen IX, stabilize this structure (Blaschke et al., 2000). However, several lines of evidence point to the possible existence of additional, hitherto unknown components of cartilage fibrils. In several instances, collagen IX is not found in cartilage fibrils. This protein could be visualized by immunoelectron microscopy only in some of the 20-nm fibrils from human fetal or neonatal cartilage, whereas a second, ultrastructurally indistinguishable fibril population appeared to be devoid of collagen IX (Bruckner et al.,

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1988). Another surprise was that cartilage of collagen IX-deficient mice is fairly normal and that the animals ¨ have mild or no skeletal abnormalities (Fassler et al., 1994; Hagg et al., 1997). Here, we identify collagen XVI as a so far unknown component of cartilage and showed that in territorial matrix, which is rich in 20-nm fibrils, collagens IX and XVI occur on similar small cartilage fibrils in a partly mutually exclusive manner. These proteins share a carboxy-terminal FACIT-domain and, therefore, the prototypic, thin cartilage fibrils may be stabilized either by collagen IX or collagen XVI. In addition, the choice of these proteins will influence the electrostatic surface charge properties of the fibrils since collagen IX often carries negatively charged glycosaminoglycan chains whereas collagen XVI does not. This provides an opportunity for modulation of interactions between the fibrils and the highly polyanionic extrafibrillar matrix rich in aggrecan and hyaluronan. Near the boundaries of the territorial and interterritorial matrix domains, i.e., at locations remote from chondrocytes, small fibrils are occasionally seen to merge into thick, strongly banded fibrils. Thus, large fibrils in the interterritorial matrix are more likely to arise by fusion of 20-nm fibrils rather than by accretion of further collagen molecules. Some of the domains of the minor collagen IX, and probably also of collagen XVI, protrude from the fibril surfaces, thereby, preventing lateral growth of 20-nm fibrils (Vaughan et al., 1988). Thick, well banded fibrils of the interterritorial matrix contain neither collagen IX nor collagen XVI but maintain a polyanionic surface by inclusion of the proteoglycan decorin (Hagg et al., 1998). Removal of collagens IX and XVI is, thus, likely to be necessary for lateral growth of cartilage fibrils beyond 20 nm. In the skin, the different fibrillar networks in the matrix accommodate diverse functions, such as providing resistance to external frictional forces or maintaining elasticity. The microfibrillar system in conjunction with elastic fibres secures elastic properties of skin. In the papillary dermis, the microfibrils insert perpendicularly into the basement membrane and extend into the dermis, where they gradually merge with the elastic fibres to form a plexus parallel to the DEJ. These microfibrils exhibit a relatively uniform diameter of 10–12 nm and display no clear banding pattern. Little is known about the composition, assembly and organization of these particular microfibrils. The best characterized molecular components are fibrillin-1 and -2 (Kielty and Shuttleworth, 1997) although other structurally similar molecules, such as MAGP 1 and 2, are also known to contribute to the structure of the fibrils (Segade et al., 2002). Here, we identified collagen XVI as a component of the microfibrillar apparatus of the skin, thus, extending the spectrum of its molecular constituents. Due to the large number of suprastructural components in this tissue, the organization and stabilization of

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the dermal ECM are subject to a particularly complex interplay between the components. Microfibrils associate with several matrix components which, in turn, are likely to interact with dermal fibrils (Raghunath et al., 1998). In this regard, the functional role of collagen XVI as a microfibrillar component may be to stabilize interactions of microfibrils with other matrix components. Collagen XVI is exclusively integrated into specialized microfibrils in the papillary dermis close to the DEJ, which do not overtly contain amorphous elastin cores. This localization may indicate that collagen XVI plays an active role in anchoring microfibrils to basement membranes, which occur not only in skin but also in many other tissues. In contrast, collagen XVI is absent from the microfibrils in the deeper reticular dermis. This distribution pattern is contrary to that of, e.g. fibulin-2, a protein with multiple binding partners which is not associated with the microfibrils immediately adjacent to the DEJ, but with the microfibrils in the deeper dermis (Raghunath et al., 1999). This observation implies that structurally distinct microfibril populations with different functional properties exist in the skin analogously to the banded cartilage fibril populations. There is no evidence for the existence of different isoforms of collagen XVI in cartilage and skin. Cloning and biosynthetic analysis of collagen XVI from different sources has yielded similar results suggesting that only ¨ one form of this collagen is found in the tissues (Grassel et al., 1996; Pan et al., 1992; Tillet et al., 1995; Yamaguchi et al., 1992). Therefore, the molecule must contain in its structure, the information for suprastructural assemblies with different partners, i.e. for the polymerization into thin cartilage fibrils or into dermal microfibrils. In vitro co-polymerization of recombinant collagen XVI with different matrix components will shed light onto its precise role in the morphogenesis of distinct suprastructures in different tissues. 4. Materials and methods 4.1. Antibodies Primary antibodies: Anti-collagen XVI antiserum (NC11) 1029; affinity-purified anti-NC11 antibody Eq (Tillet et al., 1995); anti-collagen IX monoclonal antibodies D1-9 and B3-1 (DSHB, University of Iowa); anti-fibrillin-1 monoclonal antibody Mab 69 and Mab 201 (Dr. Lynn Y. Sakai, Shriners Hospital for Children, Portland, OR); monoclonal anti-decorin antibody (Schmidt et al., 1987), monoclonal anti-collagen I antibody Mab 1340 (Chemicon International). Secondary antibodies: Fluorescein (FITC)-conjugated affiniPure goat anti-mouse IgG (Jackson ImmunoResearch); Rhodamine (TRITC)-conjugated affiniPure goat anti-rabbit IgG (Roche); horseradish-peroxidase conjugated goat anti-rabbit IgG (Kirkegard); goat anti-

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mouse IgG and IgM conjugated with 12-nm gold particles (Jackson Immuno Research); goat anti-rabbit IgG conjugated with 18-nm gold particles (Jackson Immuno Research). For immunogold electron microscopy of ultrathin sections gold particles with a diameter of 16 nm were prepared for the labeling of the primary antibodies and 8-nm gold particles for the secondary rabbit anti-mouse IgG antibodies (solid-phase absorbed against human proteins) (Medac).

in 2% not-fat milk powder in TBS–T at 4 8C overnight. The membrane was washed twice in TBS–T, once in TBSy0.5% Tween 20 and once in TBS followed by incubation with secondary goat anti-rabbit conjugated peroxidase IgG in 2% non-fat milk powder in TBS–T for 1.5 h at RT. After washing as before signal detection was performed with the ECL-Plus system (Amersham Biosciences).

4.2. Tissues

Prior to RT-PCR total RNA, extracted from chondrocytes freshly isolated from human articular cartilage as ¨ et al., 1998) and from cultured described earlier (Szuts primary human fibroblasts (RNeasy-method, Qiagen) was treated with 10 units RNase B free DNase (Roche Diagnostics GmbH) for 30 min at 37 8C. For the reverse transcription, 0.7 mg RNA and 300 pmol of Oligo (dt)15 primers (MWG Biotech, Germany) were dissolved in 41.5 ml of H20 (treated with diethylene pyrocarbonate), denaturated at 70 8C for 5 min and cooled down to RT for additional 10 min. 5 ml tenfold concentrated RT buffer (Stratagene), 1 ml of 100 mM dNTP-mix (Eppendorf), 45 units of RNase-inhibitor (30 Uyml, Eppendorf) and 50 units of Stratascript moloney murine leukemia virus reverse transcriptase (50 Uyml, Stratagene) were combined with the RNA solution to give a final volume of 50 ml. The RT-reaction was allowed to continue for 1 h at 42 8C and stopped by heating at 95 8C for 5 min. For PCR, 2 ml cDNA was supplemented with 5 ml 10= PCR-buffer, 3 ml 50 mM MgCl2, 2 ml dNTP blend (2.5 mM of each dCTP, dATP, dGTP, dTTP), 1.0 ml specific primer each (10 pmolyml), 0.3 ml Ampli-Taqpolymerase (5 Uyml; all reagents Invitrogen) and was adjusted with H2O to a final volume of 50 ml. Cycling conditions were 5 min at 95 8C for the first cycle, and 30 s at 94 8C, 1 min at 66 8C and 1 min at 72 8C for 35 subsequent cycles using a DNA-thermocycler (Biozym).

Normal human skin obtained from a local hospital for plastic surgery (Hornheide) was used for preparing fibrillar extracts for immunogold electron microscopy. Normal human skin obtained from biopsies was used for immunohistochemistry and for immunogold electron microscopic studies. Human juvenile, fetal costal and normal adult knee joint cartilage was used for polymerase chain reaction with reverse transcribed mRNA (RTPCR), western blotting, immunohistochemistry and immunogold electron microscopy of fibrillar extracts. 4.3. Western blotting Human knee joint cartilage was stored at y80 8C after surgical removal. Immediately after removal from y80 8C freezer, samples were washed in protease inhibitor cocktail (Complete Tablets, 25= concentrated, Roche Diagnostics GmbH) and after removal of adherent soft tissue approximately 280 mg of cartilage was smashed and pulverized in a mortar under liquid nitrogen. The powder was boiled in 500 ml extraction buffer containing 0.1 M Tris, 0.2 M NaCl, 2% SDS and 50 mM DTT at pH 7.6 on a Bunsen burner, the supernatant was collected and the containing protein was obtained by ethanol precipitation. Protein pellets were dissolved in phosphate buffered saline (PBS) and digested with ABC-chondroitinase (Seikagaku) and Endo-b-Galactosidase (Roche Diagnostics GmbH) for 3 h at 37 8C. Epidermolyzed human skin samples were shock frozen in liquid nitrogen and 120 mg of the upper dermis was treated as cartilage samples above. Proteins were obtained by TCA precipitation. For SDS-gel electrophoresis (SDS-PAGE) all samples were dissolved in SDS-loading buffer (0.125 M Tris– HCl, pH 6.8, 2% SDS, 50 mM DDT, 15% glycerol, 0.1% bromophenol blue) and subjected to electrophoresis on 4.5–15% polyacrylamide gradient gels. After transferring extracts onto a nitrocellulose filter (Schleicher and Schuell), the membrane was treated with 5% non-fat milk powder in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS–T) for 1.5 h at room temperature (RT). After rinsing twice with TBS–T, the membrane was incubated with antibody Eq (1:1000)

5. RT-PCR

5.1. Primer sequences

GAPDH Collagen XVI

59-CTG ACT TCA ACA GCG ACA CC-39 59-CCC TGT TGC TGT AGC CAA AT-39 59-CTG GTG ATC CTG TAC GAG CCA GG-39 59-CCA TCC TGA AGG TTG GAC AGG GC-39

5.2. Fibrillar extracts prepared from skin and cartilage 5.2.1. Dermal extracts Normal human skin was obtained from a local hospital for plastic surgery and stored immediately in PBS at y 80 8C. Samples of approximately 2 cm2 were thawed

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and stirred overnight at 4 8C in epidermolysis buffer (20 mM ethylenediamine tetra-acetic acid (EDTA), 50 mM Tris; pH 7.4), containing a mixture of protease inhibitors (Pepstatin, Leupeptin, Pefabloc, Aprotinin, each concentration at 2 mgyml). Subsequent addition of solid sodium chloride up to a final concentration of 1 M was followed by 2–3 h of stirring, thus, allowing the epidermis to be easily stripped off the dermis according to Bruckner-Tuderman et al. (1987). Samples were either ready to be used directly after epidermolysis or to be stored in PBS at y80 8C. Epidermolyzed skin-samples were frozen onto a metal surface using liquid nitrogen. The upper 200 mm of the dermal samples were planed off with a dermatome (Aeskulap) and while still frozen were cut into small pieces with a scalpel and suspended in a homogenization buffer (2 mM sodium phosphate; 150 mM sodium chloride, pH 7.4) containing a mixture of protease inhibitors (50 mM 6-aminohexanoic acid; 5 mM benzamidin; 5 mM N-ethylmaleimide (NEM); 0.1 mM phenylmethylsulfonyl fluoride (PMSF)). The samples were repeatedly homogenized on ice with a Polytron (Kinematica) and centrifuged between each homogenization step for 3 min at 2061=g and 4 8C. The supernatants containing the fibrillar fragments received after the second and third centrifugation step were directly used for adsorption step to grids for analysis in electron microscopy. 5.2.2. Cartilage extracts Juvenile costal or normal adult knee cartilage slices were homogenized with a Polytron (Kinematica) in 15 volumes of 0.15 M sodium chloride, 2 mM sodium phosphate, pH 7.4 containing a mixture of proteinase inhibitors (0.1 mM PMSF, 0.1 M 6-aminohexanoic acid, 5 mM benzamidine, 5 mM NEM and 20 mM EDTA). The obtained suspension was centrifuged at 27 000=g for 30 min at 4 8C and the clear supernatant was collected. This procedure was repeated twice with fresh extraction buffer and the three supernatants were combined. Partial purification and concentration of the obtained fibril fragments was achieved by high-speed centrifugation of these extracts at 115 000=g for 2 h at 4 8C. The final pellet was redissolved in PBS and adsorbed to grids as described above. 5.3. Immunohistochemistry and confocal microscopy Cryosections (4 mm) of fetal human costal cartilage and normal human skin were air-dried, fixed in acetone at 4 8C and rehydrated in PBS. Cartilage sections only were incubated for 90 min with 0.05% hyaluronidase from bovine testes (359 Uymg, Sigma) at 37 8C. Blocking both, skin and cartilage specimen in 5% normal goat serum containing 1% bovine serum albumin (BSA) and protease inhibitors (Complete Mini tablets

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1=, Roche Diagnostics GmbH) overnight at 4 8C was followed by washing with PBS. Sections were incubated with a mixture of Mab 69 diluted 1:50 or 1:100 and Eq diluted 1:100 in PBS containing 1% BSA and protease inhibitors for 7 h at 4 8C. After washing with PBS the tissues were incubated with a mixture of goat anti-mouse FITC IgG diluted 1:100 and goat anti-rabbit TRITC IgG diluted 1:250 in PBS containing 1% BSA and protease inhibitors for 1 h at RT. After rinsing with PBS and 0.05% Tween 20 the sections were mounted in Fluoromount (Southern BioTechnology) and analyzed with a Nikon Eclipse E 600 confocal laser scanning microscope (Nikon). 5.4. Immunoelectron microscopy of fibrillar extracts For immunoelectron-microscopy aliquots of fibrillar extracts from cartilage or skin were spotted onto sheets of Parafilm. Copper grids coated with Formvarycarbon were floated on the drops for 5 min to allow adsorption of fibril fragments. The grids were subsequently washed with PBS, and treated for 30 min with 2% (wt.yvol.) BSA in PBS. For the localization of collagen IX positive fibrils in cartilage fibril fragments adsorbed to Formvary carbon coated nickel grids were treated for 30 min at 37 8C with hyaluronidase from bovine testes (359 Uy mg, Sigma) and were subsequently washed several times with PBS. The adsorbed material was allowed to react for 2 h with an antibody to collagen XVI (Eq) and antibodies to fibrillin (Mab 201 or Mab 69) diluted 1:100 or a mixture of antibodies to collagen IX (D1-9 and B3-1) diluted 1:100 in PBS containing 0.2% Aurion BSA-c (Biotrend) and 0.1% Tween 20 (blocking solution). After washing five times with blocking solution, the grids were put on drops of blocking solution containing goat antibodies directed against rabbit or mouse immunoglobulins conjugated to colloidal gold particles (diluted 1:30) (Jackson Immuno Research). For double labeling experiments, a mixture of gold particles of two different sizes (12 and 18 nm) was used. Finally, the grids were washed five times with distilled water, and negatively stained with 2% uranyl acetate (Merck) for 10 min. Control experiments were done with the first antibody omitted. Electron micrographs were taken at 60 kV with an EM 410 electron microscope (Philips). 5.5. Immunoelectron microscopy of ultrathin tissue sections 5.5.1. Tissue samples Adult articular cartilage was obtained during autopsy from the lateral femoral condyles of knee joints as was abdominal skin taken from three 34-, 44- and 54-yearold male accident victims within 6 h post-mortem. In no case did the cartilage or skin tissue show any histological sign of degenerative changes, the deceased

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had been suffering from neither systemic disease nor other health impairment. 5.5.2. Tissue embedding and sectioning Tissue samples were cut into 1=1 mm2 pieces, fixed immediately for 30 min with 4% paraformaldehyde and 0.5% glutaraldehyde in 0.15 M PBS (pH 7.4), dehydrated in up to 70% ethanol and embedded in the hydrophilic resin LR-Gold䉸 (London Resin Company). Semithin (1 mm) sections for the histopathological evaluation and ultrathin (70 nm) sections for the ultrastructural analysis were cut. The sectioning plane was determined and if necessary re-embedding guaranteed a longitudinal orientation of the specimens. 5.5.3. Electron microscopic immunogold-histochemistry Ultrathin tissue sections on nickel grids were incubated with the 16-nm gold labeled anti-collagen XVI (Eq ; 1:200, 1 h at RT) or the anti-fibrillin-1 antibody (Mab 69; 1:20, 1 h at RT). For the localization of collagen IX, chondroitinase ABC (Sigma) treatment was followed by incubation with the anti-collagen type IX antibodies (both 1:100, 1 h at RT). The grids were thoroughly rinsed with PBS and the rabbit anti-mouse antibody (1:300 for 20 min. at RT) was applied, followed by rinsing with water and contrasting with uranyl acetate and lead citrate (each for 10 min). Subsequently, grids were examined and micrographs taken up to a magnification of 21 000= with an electron microscope Leo 906 E. For controls, tissue sections were incubated with pure colloidal gold solution in order to exclude binding of uncoupled gold particles to tissue structures. To exclude non-specific IgG binding, the reaction was performed with the gold-coupled rabbit anti-mouse IgG and serum of non-immunized animals. 5.5.4. Double labeling For double labeling, two of the following antibodies either Eq, Mab 69 or D1-9 were coupled to gold particles of differing sizes (16 and 8 nm) were incubated consecutively under the conditions described above, starting with the larger particle size. The remainder of the procedure was the same as described above. Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (Grants: Gr 1301y2-1 and 2-2; Re 1021y3-1 and 4-1; SFB 492yA2 and SFB 492yA3) and by a Lise-Meitner-Fellowship (821-6037) assigned to ¨ Susanne Grassel. The NC-11 antibody Eq was a generous gift from Dr. R. Timpl, MPI, Martinsried, Germany. The monoclonal antibodies against human fibrillin-1 were generously provided by Dr. Lynn Y. Sakai, Shriners Hospital for Children, Portland, Oregon. The monoclonal antibody against decorin was a friendly

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