Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components.

Transforming growth factor (TGF)-beta-induced chondrogenesis of mesenchymal stem cells derived from bone marrow involves the rapid deposition of a cartilage-specific extracellular matrix. The sequential events in this pathway leading from the undifferentiated stem cell to a mature chondrocyte were investigated by analysis of key matrix elements. Differentiation was rapidly induced in cells cultured in the presence of TGF-beta 3 or -beta 2 and was accompanied by the early expression of fibromodulin and cartilage oligomeric matrix protein. An increase in aggrecan and versican core protein synthesis defined an intermediate stage, which also involved the small leucine-rich proteoglycans decorin and biglycan. This was followed by the appearance of type II collagen and chondroadherin. The pathway was also characterized by the appearance of type X collagen, usually associated with hypertrophic cartilage. There was also a change in the pattern of sulfation of chondroitin sulfate, with a progressive increase in the proportion of 6-sulfated species. The major proportion of newly synthesized glycosaminoglycan was part of an aggregating proteoglycan network. These data allow us to define the phenotype of the differentiated cell and to understand in greater detail the sequential process of matrix assembly.

The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an epitope on endoglin (CD105).

Mesenchymal stem cells are multipotent cells resident in the bone marrow throughout adulthood which have the capacity to differentiate into cartilage, bone, fat, muscle, and tendon. A number of monoclonal antibodies raised against human MSCs have been shown to react with surface antigens on these cells in vitro. A protein of molecular mass 92 kDa was immunoprecipitated using the SH-2 monoclonal antibody. This was purified and identified by peptide sequencing analysis and mass spectrometry as endoglin (CD105), the TGF-beta receptor III present on endothelial cells, syncytiotrophoblasts, macrophages, and connective tissue stromal cells. Endoglin on MSCs potentially plays a role in TGF-beta signalling in the control of chondrogenic differentiation of MSCs and also in mediating interactions between MSCs and haematopoietic cells in the bone marrow microenvironment.

Mesenchymal stem cell surface antigen SB-10 corresponds to activated leukocyte cell adhesion molecule and is involved in osteogenic differentiation.

Bone marrow contains a rare population of mesenchymal stem cells (MSCs) capable of giving rise to multiple mesodermal tissues including bone, cartilage, tendon, muscle, and fat. The cell surface antigen recognized by monoclonal antibody SB-10 is expressed on human MSCs but is lost during their developmental progression into differentiated phenotypes. Here we report on the immunopurification of the SB-10 antigen and its identification as activated leukocyte-cell adhesion molecule (ALCAM). Mass spectrometry establishes that the molecular mass of ALCAM is 80,303 +/- 193 Da and that it possesses 17,763 +/- 237 Da of N-linked oligosaccharide substituents. Molecular cloning of a full-length cDNA from a MSC expression library demonstrates nucleotide sequence identity with ALCAM. We also identified ALCAM homologs in rat, rabbit, and canine MSCs, each of which is over 90% identical to human ALCAM in their peptide sequence. The addition of antibody SB-10 Fab fragments to human MSCs undergoing osteogenic differentiation in vitro accelerated the process, thereby implicating a role for ALCAM during bone morphogenesis and adding ALCAM to the group of cell adhesion molecules involved in osteogenesis. Together, these results provide evidence that ALCAM plays a critical role in the differentiation of mesenchymal tissues in multiple species across the phylogenetic tree.

Post-translational modifications in cartilage oligomeric matrix protein. Characterization of the N-linked oligosaccharides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Analysis of the carboxymethylated subunit of human cartilage oligomeric matrix protein (COMP) by matrix-assisted laser desorption time-of-flight mass spectrometry indicated a protonated molecular mass of 86949 +/- 149 Da, compared with 83547.0 Da calculated from the sequence. Treatment with N-glycanase caused a reduction in mass of 3571 +/- 219 Da, but there was no loss of mass after treatment with O-glycanase or neuraminidase. Peptides containing two putative sites of N-glycosylation were purified and characterized. Analysis of the masses of these after N-glycanase treatment indicated that one was substituted at Asn-101 with an oligosaccharide of mass 1847. 2 +/- 6.6 Da, and the other was unsubstituted at Asn-124. The remaining site of attachment, at Asn-721, was, therefore, also substituted with an oligosaccharide of mass 1724 +/- 226 Da. Analysis of the total monosaccharide content by chemical methods indicated that there were no additional oligosaccharide substituents. The MALDI-TOF mass spectra of COMP from bovine fetal and adult cartilage were compared, indicating a more heterogeneous pattern of substitution at Asn-101 in the fetal form. Since COMP is distributed throughout the pericellular and territorial environments in developing cartilage but occupies the interterritorial zone in mature cartilage, these changes in glycosylation may allow for different intermolecular interactions.

The structure of a 38-kDa leucine-rich protein (chondroadherin) isolated from bovine cartilage.

A leucine-rich protein, chondroadherin, has been isolated from dissociative extracts of articular cartilage, and its primary structure has been determined by both direct protein sequencing and DNA sequence analysis of polymerase chain reaction products and cDNA clones. This protein is identical to the 36-kDa protein which was isolated by Larsson et al. (Larsson, T., Sommarin, Y., Paulsson, M., Antonsson, P., Hedbom, E., Wendel, M., and Heinegård, D. (1991) J. Biol. Chem. 266, 20428-20433). It has 337 amino acids and exists in several isoforms. The two major isoforms are a form with a calculated molecular weight of 38,353 and a pI of 9.76 and a smaller form with a calculated molecular weight of 37,304 and a pI of 9.5. The two isoforms result from a cleavage near the C terminus. A further level of heterogeneity is found in that an extra alanine can be found prior to the N-terminal cysteine. There are 9 cysteines; disulfide bonds have been directly identified between Cys282-Cys324 and Cys284-Cys304. The principal feature of the protein is a series of 10 leucine-rich repeats. The most N-terminal of these repeats contains a cysteine (Cys63) which is not disulfide-bonded and which is difficult to derivatize. It is likely that this free cysteine is involved in structure-stabilizing hydrogen bonding. The mRNA is approximately 1.6 kilobases, of which 511 base pairs is a 3'-untranslated region between the stop codon and the polyadenylation signal. Based on anchored polymerase chain reaction analysis of the mRNA, there is some minor heterogeneity in the position of the 5' end of the message.

Analysis of the catabolism of aggrecan in cartilage explants by quantitation of peptides from the three globular domains.

A method has been developed for the production, isolation, and quantitation of 15 marker peptides from the three globular domains (G1, G2, and G3) and the interglobular domain of bovine aggrecan (aggregating cartilage proteoglycan). Three of the peptides are from G1, two are from the interglobular domain, four are from G2, and six are from G3. The method involves separation of tryptic peptides by sequential anion-exchange, cation-exchange, and reversed-phase high performance liquid chromatography and quantitation by absorbance at 220 nm. The values obtained (peak area per microgram of core protein) were a function of the molar yield and also the size and aromatic residue content of individual peptides. This procedure has been applied to aggrecan purified from fresh calf articular cartilage and to aggrecan isolated from the medium and tissue compartments of cartilage explant cultures, maintained in basal medium for 15 days without and with interleukin-1 alpha. These analyses indicate that aggrecan which is released into explant medium has a reduced content of the G1 domain, but has a normal content of the G2 domain, the COOH-terminal region of the interglobular domain, and also the G3 domain. On the other hand, aggrecan which is retained by the cartilage during 15 days of culture has a normal content of G1, interglobular domain, and G2 domains, but, in the presence of interleukin-1 alpha, it has a reduced content of the G3 domain. The percentage of medium molecules which retained the G1 domain was higher in control cultures (about 35%) than in interleukin cultures (about 20%), and this was consistent with the relative aggregability of these samples. Taken together these results suggest that catabolism of aggrecan in articular cartilage involves a specific proteolysis of the core protein at a site which is within the interglobular domain and NH2-terminal to the sequence LPGG. This process occurs in control cultures but is accelerated by the addition of interleukin-1 alpha. Degraded molecules which lack the G1 domain are released preferentially into the medium; however, these molecules carry both the G2 and G3 domains, indicating that these domains do not confer strong matrix binding properties on aggrecan. The method described here for the isolation of peptides from bovine aggrecan should have wide application to structural and biosynthetic studies on this molecule in species such as human and rat, since many of the marker peptides are from highly conserved regions of the aggrecan core protein.

Catabolism of aggrecan in cartilage explants. Identification of a major cleavage site within the interglobular domain.

The catabolism of aggrecan has been studied in calf articular cartilage explant cultures. The chondroitin sulfate-rich, high buoyant density products that accumulate in culture medium have been purified, and NH2-terminal sequence data have been obtained. Aggrecan released from the tissue in the presence or absence of interleukin-1 alpha, whether analyzed before or after reduction and alkylation, exhibited only one major and one minor NH2-terminal sequence. The major sequence, ARGXVILXAKPDF, shows very high similarity to a region of the interglobular domain (between the G1 and G2 domains) of both human and rat aggrecan. The minor sequence, VEVS, was that previously described for the NH2 terminus of the intact core protein. These results indicate that catabolism of aggrecan in cartilage explants involves proteolytic cleavage within a conserved region of the interglobular domain and that this results in the separation of the G1 domain from the remainder of the molecule. A major product of this process is a large nonaggregating species that consists of an NH2-terminal sequence beginning with ARG (and composed of about 100 residues of the interglobular domain) that is attached to an intact G2 domain followed by an extended section of the chondroitin sulfate-bearing domain toward the COOH terminus.

Isolation and characterization of disulfide-bonded peptides from the three globular domains of aggregating cartilage proteoglycan.

The aggregating cartilage proteoglycan core protein contains two globular domains near the N terminus (G1 and G2) and one near the C terminus (G3). The G1-G3 domains contain 10, 8, and 10 cysteine residues, respectively. The disulfide assignments of the G1 domain have previously been deduced (Neame, P. J., Christner, J. E., and Baker, J. R. (1987) J. Biol. Chem. 262, 17768-17778) as Cys1-Cys2, Cys3-Cys6, Cys4-Cys5, Cys7-Cys10, and Cys8-Cys9, in which the numbers cited after the half-cystine residues are their relative positions from the N terminus. Here we describe a method for the isolation of disulfide-bonded peptides from tryptic digests of bovine nasal cartilage monomer. Sequence analysis of these peptides has allowed us to confirm the pairings previously determined for the G1 domain and to assign a disulfide pattern for the G2 domain of Cys11-Cys14, Cys12-Cys13, Cys15-Cys18, and Cys16-Cys17, in which the Cys15-Cys18 pairing was deduced indirectly. Similarly, for the G3 domain, a pattern of Cys19-Cys20, Cys21-Cys24, Cys22-Cys23, Cys25-Cys27, and Cys26-Cys28 was assigned, in which the Cys22-Cys23 pair was deduced indirectly. The G2 domain therefore contains disulfide bonding which is characteristic of the tandem repeat structures found in the G1 domain and link protein, and the G3 domain contains the three disulfide linkages previously assigned to the family of C-type animal lectins. The method described here, which combines anion-exchange, cation-exchange, and reversed-phase chromatography, should have broad application to the isolation of disulfide-bonded peptides from other heavily glycosylated proteins and proteoglycans.