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Sub-optimal clinical outcomes after implantation of animal-derived tissue matrices may be attributed to the nature of the processing of the material or to an immune response elicited in response to xenogeneic epitopes. The ability to produce a porcine-derived graft that retains the structural integrity of the extracellular matrix and minimizes potential antigenic response to galactose-α-(1,3)-galactose terminal disaccharide (α-Gal) may allow the scaffold to support regeneration of native tissue. Dermal tissue from wild-type (WT-porcine-derived acellular dermal matrix [PADM]) or Gal-deficient (Gal−/−PADM) pigs was processed to remove cells and DNA while preserving the structural integrity of the extracellular matrix. In addition, the WT tissue was subjected to an enzymatic treatment to minimize the presence of α-Gal (Gal-reduced PADM). Extracellular matrix composition and integrity was assessed by histological, immunohistochemical (IHC), and ultrastructural analysis. In vivoperformance was evaluated by implantation into the abdominal wall of Old World primates in an exisional repair model. Anti-α-Gal activity in the serum of monkeys implanted subcutaneously was assessed by ELISA. Minimal modification to the extracellular matrix was assessed by evaluation of intact structure as demonstrated by staining patterns for type I and type VII collagens, laminin, and fibronectin similar to native porcine skin tissues. Explants from the abdominal wall showed evidence of remodeling, notably fibroblast cell repopulation and revascularization, as early as 1 month. Serum ELISA revealed an initial anti-α-Gal induction that decreased to baseline levels over time in the primates implanted with WT-PADM, whereas no or minimal anti-Gal activity was detected in the primates implanted with Gal−/−PADM or Gal-reduced PADM. The combination of a nondamaging process, successful removal of cells, and reduction of xenogeneic α-Gal antigens from the porcine dermal matrix are critical for producing a material with the ability to remodel and integrate into host tissue and ultimately support soft tissue regeneration.

Decorin, a member of the small leucine-rich proteoglycan gene family, down-regulates members of the ErbB receptor tyrosine kinase family and attenuates their signaling, leading to growth inhibition. We investigated the effects of decorin on the growth of ErbB2-overexpressing mammary carcinoma cells in comparison with AG879, an established ErbB2 kinase inhibitor. Cell proliferation and anchorage-independent growth assays showed that decorin was a potent inhibitor of breast cancer cell growth and a pro-apoptotic agent. When decorin and AG879 were used in combination, the inhibitory effect was synergistic in proliferation assays but only additive in both colony formation and apoptosis assays. Active recombinant human decorin protein core, AG879, or a combination of both was administered systemically to mice bearing orthotopic mammary carcinoma xenografts. Primary tumor growth and metabolism were reduced by approximately 50% by both decorin and AG879. However, no synergism was observed in vivo. Decorin specifically targeted the tumor cells and caused a significant reduction of ErbB2 levels in the tumor xenografts. Most importantly, systemic delivery of decorin prevented metastatic spreading to the lungs, as detected by novel species-specific DNA detection and quantitative assays. In contrast, AG879 failed to have any effect. Our data support a role for decorin as a powerful and effective therapeutic agent against breast cancer due to its inhibition of both primary tumor growth and metastatic spreading.

The outer surface protein C (OspC) is one of the major host-induced antigens of Borrelia burgdorferi, the causative agent of Lyme disease. We have solved the crystal structure of recombinant OspC to a resolution of 2.5 Å. OspC, a largely α-helical protein, is a dimer with a characteristic central four-helical bundle formed by association of the two longest helices from each subunit. OspC is very different from OspA and similar to the extracellular domain of the bacterial aspartate receptor and the variant surface glycoprotein from Trypanosoma brucei. Most of the surface-exposed residues of OspC are highly variable among different OspC isolates. The membrane proximal halves of the two long α-helices are the only conserved regions that are solvent accessible. As vaccination with recombinant OspC has been shown to elicit a protective immune response in mice, these regions are candidates for peptide-based vaccines.

Integrins α1β1and α2β1are two major collagen receptors on the surface of eukaryotic cells. Binding to collagen is primarily due to an A-domain near the N terminus of the α chains. Previously, we reported that recombinant A-domain of α1β1(α1A) had at least two affinity classes of binding sites in type I collagen (Rich, R. L., et al.(1999)J. Biol. Chem.274, 24906–24913). Here, we compared the binding of the recombinant A-domain of α2β1(α2A) to type I collagen with that of α1A using surface plasmon resonance and showed that α2A exhibited only one detectable class of binding sites in type I collagen, with a KDof ∼10 μmat ∼3 binding sites per collagen molecule. We further demonstrated that α1A and α2A competed with each other for binding to type I collagen in enzyme-linked immunosorbent assay (ELISA), suggesting that the binding sites in collagen for the two A-domains overlap or are adjacent to each other. By using rotary shadowing, the complexes of α1A- and α2A-procollagen were visualized. Morphometric analyses indicated three major binding regions (near the N terminus, in the central part, and near the C terminus) along the type I procollagen molecule for both A-domains. The positions of the respective binding regions for α1A and α2A were overlapping with or adjacent to each other, consistent with the ELISA results. Analysis of the sequences of type I collagen revealed that GER or GER-like motifs are present at each of the binding regions, and notably, the central region contains the GFOGER sequence, which was previously identified as a high affinity site for both α1A and α2A (Knight, C. G., et al.(2000) J. Biol. Chem.275, 35–40). Peptides containing GLOGERGRO (peptide I, near the N terminus), GFOGERGVQ (peptide II, central), and GASGERGPO (peptide III, near the C terminus) were synthesized. Peptides I and II effectively inhibited the binding of α1A and α2A to type I collagen, while peptide III did so moderately. The N-terminal site in type I collagen has the sequence GLOGER in all three chains. Thus, it seems that peptide I represents a newly discovered native high affinity site for α1A and α2A.

A putative collagen-binding MSCRAMM, Ace, of Enterococcus faecaliswas identified by searching bacterial genome data bases for proteins containing domains homologous to the ligand-binding region of Cna, the collagen-binding MSCRAMM from Staphylococcus aureus. Ace was predicted to have a molecular mass of 71 kDa and contains features characteristic of cell surface proteins on Gram-positive bacteria, including a LP XTG motif for cross-linking to the cell wall. The N-terminal region of Ace contained a region (residues 174–319) in which 56% of the residues are identical or similar when compared with the minimal ligand-binding region of Cna (Cna 151–318); the remainder of the Ace A domain has 46% similarity with the corresponding region of the Cna A domain. Antibodies raised against recombinant Ace A domain were used to verify the cell surface expression of Ace on E. faecalis. These antibodies also effectively inhibited the adhesion of enterococcal cells to a collagen substrate, suggesting that Ace is a functional collagen-binding MSCRAMM. Structural modeling of the conserved region in Ace (residues 174–319) suggested a structure very similar to that reported for residues 151–318 of the Cna collagen-binding domain in which the ligand-binding site was identified as a trench transversing a β-sheet face (Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol.10, 833–838). Biochemical analyses of recombinant Ace and Cna A domains supported the modeling data in that the secondary structures were similar as determined by CD spectroscopy and both proteins bound at multiple sites in type I collagen with micromolar affinities, but with different apparent kinetics. We conclude that Ace is a collagen-binding MSCRAMM on enterococci and is structurally and functionally related to the staphylococcal Cna protein.

Most mammalian cells and some pathogenic bacteria are capable of adhering to collagenous substrates in processes mediated by specific cell surface adherence molecules. Crystal structures of collagen-binding regions of the human integrin α2β1and a Staphylococcus aureusadhesin reveal a “trench” on the surface of both of these proteins. This trench can accommodate a collagen triple-helical structure and presumably represents the ligand-binding site (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem.272, 28512–28517; Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., Schneider, A., DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol.4, 833–838). We report here the crystal structure of the α subunit I domain from the α1β1integrin. This collagen-binding protein also contains a trench on one face in which the collagen triple helix may be docked. Furthermore, we compare the collagen-binding mechanisms of the human α1integrin I domain and the A domain from the S. aureuscollagen adhesin, Cna. Although the S. aureusand human proteins have unrelated amino acid sequences, secondary structure composition, and cation requirements for effective ligand binding, both proteins bind at multiple sites within one collagen molecule, with the sites in collagen varying in their affinity for the adherence molecule. We propose that (i) these evolutionarily dissimilar adherence proteins recognize collagen via similar mechanisms, (ii) the multisite, multiclass protein/ligand interactions observed in these two systems result from a binding-site trench, and (iii) this unusual binding mechanism may be thematic for proteins binding extended, rigid ligands that contain repeating structural motifs.

Proteoglycan production was examined in cultures of thioglycollate‐elicited peritoneal macrophages obtained from White Carneau and Show Racer pigeons. Following a 24‐h incubation in the presence of [35S]sulfate and [3H]serine, total production and distribution of 35S‐labeled proteoglycan into media (60–65%), pericellular (21–27%), and intracellular (13–14%) compartments was similar in White Carneau and Show Racer macrophage cultures. Media proteoglycans consisted of high‐molecular‐weight chondroitin sulfate proteoglycan, low‐molecular‐weight chondroitin sulfate proteoglycan, and heparan sulfate proteoglycan. High‐molecular‐weight chondroitin sulfate proteoglycan was predominantly 6‐sulfated (80%) and contained a core protein larger than 200 kd, whereas low‐molecular‐weight chondroitin sulfate proteoglycan was 4‐sulfated and contained a 28‐kd core protein. Pericellular proteoglycan was similar in size to low‐molecular‐weight proteoglycan and consisted of a predominantly 6‐sulfated (75%) chondroitin sulfate proteoglycan and heparan sulfate proteoglycan. Intracellular 35S‐labeled chondroitin sulfate and heparan sulfate were smaller than media and pericellular proteoglycans, suggestive of intracellular degradative processing.

Recombinant constructs encoding the fibrinogen‐binding domains of ClfA and ClfB from Staphylococcus aureushave been crystallized. ClfA was crystallized in the orthorhombic space group P212121with unit‐cell parameters a= 39.58, b= 81.39 and c= 112.65 Å. A complete data set was recorded to 2.1 Å resolution and had a Vmof 2.3 Å3Da−1with 46.5% solvent, suggesting one molecule per asymmetric unit. Co‐crystals of ClfA with the 17 amino‐acid C‐­terminal peptide of fibrinogen γ‐chain diffracted to 2.1 Å resolution and had unit‐cell parameters a= 39.11, b= 81.39 and c= 109.51 Å in the space group P212121. ClfB was crystallized in the tetragonal space group P41212 or P43212 with unit‐cell parameters a= 96.31, b= 96.31 and c= 84.13 Å and diffracted to 2.45 Å resolution. The estimated Vmof 2.6 Å3Da−1with 53% solvent indicated one molecule in the asymmetric unit.

Recombinant proteins of monomeric and dimeric B‐domain repeats of a Staphylococcus aureusFDA 574 collagen‐binding adhesin have been crystallized. The single repeat unit (B1) was crystallized in a body‐centered orthorhombic lattice with a= 96.9, b= 101.3, c= 120.8 Å in either the I222 or I212121space group. These crystals diffracted to 2.5 Å resolution and the calculated Vmvalues of 3.2 and 2.2 Å3Da−1suggest the possibility of a dimer or a trimer in the asymmetric unit. The two‐repeat fragment (B1B2) crystallized in the orthorhombic space group P212121with cell dimensions a= 42.4, b= 79.4, c= 130.4 Å and diffracted to 2.3 Å resolution. For this species, the calculated Vmvalue of 2.2 Å3Da−1indicates the presence of a monomer in the asymmetric unit.