The effect of link peptide on proteoglycan synthesis in equine articular cartilage.

The basal rate of in vitro proteoglycan (PG) synthesis in explants of equine articular cartilage was subject to considerable variation in animals of the same age but was greater in younger than older animals. Synthesis of PGs in explant cultures was stimulated by a synthetic link peptide, identical in sequence to the N-terminus of the link protein (LP) of PG aggregates, in a similar manner to that demonstrated previously for human articular cartilage [Biochem. Soc. Trans. 25 (1997) 427; Arthritis Rheum. 41 (1998) 157]. Stimulation occurred in tissue from animals ranging from 1 to 30 years old but older animals required higher concentrations of peptide to produce a measurable response. Synthesis of PGs increased in a concentration-dependent manner and was paralleled by increases in the ability of aggrecan monomers to form aggregates with hyaluronan (HA). In addition to its effect on synthesis of PGs, link peptide also increased synthesis of both aggrecan and LP mRNA. Cartilage explant and chondrocyte cultures secreted small amounts of biologically active interleukin 1 (IL 1) and secretion of this cytokine was reduced considerably by the addition of link peptide. Reduction in the activity of this catabolic cytokine coupled with the increased synthesis of mRNA for aggrecan and link peptide may be the mechanism by which link peptide exerts its positive effect on the rate of PG synthesis in articular cartilage.

Link peptide cartilage growth factor is degraded by membrane proteinases.

The peptide DHLSDNYTLDHDRAIH (Link N), cleaved from the N-terminus of the link protein component of cartilage proteoglycan aggregates by the action of stromelysin, can act as a growth factor and stimulate synthesis of proteoglycans and collagen in articular cartilage [McKenna, Liu, Sansom and Dean (1998) Arthritis Rheum. 41, 157-161]. The mechanism by which this biologically active peptide is degraded and inactivated was investigated using U937 monocytes as a model cell. Time-course experiments showed that two major proteases, an initial serine proteinase followed by a metalloproteinase, acted in sequence. Analysis of the resulting fragments showed that the serine endopeptidase cleavage was at the Leu(3)-Ser(4) bond to produce the peptide SDNYTLDHDRAIH. The terminal serine could then be removed from the resulting peptide by an aminopeptidase. A second metallopeptidase liberated the peptides SDNYTL or DNYTL from DHDRAIH by cleavage at the Leu(9)-Asp(10) bond. The DNYTL peptide intermediate was degraded too rapidly to allow sequencing and sequential aminopeptidase cleavages removed further amino acids from the N-terminus of the remaining DHDRAIH peptide. The identical patterns of breakdown that occurred when either whole cells or purified plasma membranes were used indicated that proteolysis and inactivation of Link N was carried out entirely by membrane-associated enzymes.

An N-terminal peptide from link protein can stimulate biosynthesis of collagen by human articular cartilage.

Previous studies have shown that a peptide identical in sequence to the N-terminal of link protein can function as a growth factor and up-regulate proteoglycan synthesis by human articular cartilage in explant culture (L. A. McKenna et al., Arthritis Rheum. 41, 157-162, 1998). The present study has extended these investigations to determine the effects of this peptide on the synthesis of collagen, another essential component of normal cartilage matrix. Explants from normal adult knee cartilage were maintained for periods of up to 8 days in medium with or without serum. Peptides were added during each day of culture. Synthesis of collagen was determined by the incorporation of [3H]proline into hydroxyproline and proteoglycans by incorporation of [35S]sulfate. The type of newly synthesized collagen was measured by SDS-polyacrylamide gel electrophoresis, fluorography, and immunoblotting. The link protein peptide stimulated synthesis of type II collagen in cartilage from a number of different subjects. Maximum up-regulation of synthesis was attained at a concentration of 100 ng/ml, similar to that observed previously for up-regulation of proteoglycan. Synthesis was up-regulated in both the presence and the absence of serum, although the overall rate of synthesis was greater when serum was added. The findings that this link peptide growth factor stimulated synthesis of proteins, including collagen, in a manner analogous to that shown previously for proteoglycans support the hypothesis that this peptide may have an important role in the feedback control of cartilage matrix synthesis.

The macromolecular characteristics of cartilage proteoglycans do not change when synthesis is up-regulated by link protein peptide.

Previous studies have shown that a synthetic, unglycosylated analogue of the N-terminal peptide from link protein can function as a growth factor and up-regulate proteoglycan biosynthesis in explant cultures of normal human articular cartilage from a wide age range of subjects (McKenna et al., Arthritis Rheum. 41 (1998) 157-162). The present work further shows that link peptide increased proteoglycan synthesis by cartilage cultured in both the presence and absence of serum, suggesting that the mechanism of up-regulation may be different from that of insulin-like growth factors. The proteoglycans synthesised during stimulation with link peptide were of normal hydrodynamic size and the ratio of core protein to glycosaminoglycan side chains and the proportions of the large proteoglycan aggrecan to the small proteoglycans, decorin and biglycan, remained constant. Aggrecan molecules were equally capable of forming aggregates as those from control tissues and the relative proportions of decorin and biglycan were unchanged showing that both were co-ordinately up-regulated. These results confirmed that this novel peptide is a potent stimulator of proteoglycan synthesis by articular cartilage and showed that the newly synthesised proteoglycans were of normal composition.

Re-expression of differentiated proteoglycan phenotype by dedifferentiated human chondrocytes during culture in alginate beads.

The proteoglycans (PGs) synthesised by normal human articular chondrocytes and a chondrocyte cell line cultured in monolayer and alginate beads were compared. Chondrocytes became dedifferentiated after serial subcultures in monolayer, exhibited a fibroblastic morphology and synthesised a large proportion of lower molecular weight, dermatan sulphate containing PGs. When transferred into alginate beads, the cells quickly regained their spherical shape and actively incorporated [3H]thymidine and [35S]sulphate during 70 days of culture. This resulted in a continuous increase in their DNA content and a rapid deposition of PGs for the first 25 days of culture, which then remained stable. Immediately after dedifferentiated chondrocytes were encapsulated into alginate beads, they began to synthesise a population of PGs with normal monomer size and an increased ability to form aggregates. The monomer size of newly synthesised PGs remained unchanged during extended periods of culture, but their ability to form aggregates and the ratios of chondroitin-6-sulphate to chondroitin-4-sulphate in their glycosaminoglycan chains gradually increased for the first 25 days before reaching normal values. Parallel experiments with HCS-2/8 cells, derived from a human chondrosarcoma, showed that they followed a similar pattern of development in alginate culture. The ability of their newly synthesised PGs to form aggregates increased with time and their sulphation pattern also gradually became normal. These results showed that culture in alginate promoted redifferentiation of dedifferentiated articular chondrocytes and assisted differentiation of HCS-2/8 chondrocytes. However, complete redifferentiation took a period of several weeks, after which synthesis of normal aggregating PGs was maintained.

An N-terminal peptide from link protein stimulates proteoglycan biosynthesis in human articular cartilage in vitro.

OBJECTIVE: To determine the effects of a synthetic N-terminal peptide from link protein on the synthesis of proteoglycans by human articular cartilage. METHODS: Explants from adult knee cartilage were maintained for 4 days in serum-free Dulbecco's modified Eagle's medium. Peptides were added for the final 2 days of culture. Synthesis of proteoglycans and proteins was measured by the incorporation of 35S-sulfate and 3H-serine. The sizes, sulfation patterns, and serine: sulfate ratios of newly synthesized glycosaminoglycans were measured by gel chromatography, high performance liquid chromatography, and ion-exchange chromatography. RESULTS: The N-terminal peptide stimulated proteoglycan synthesis in cartilage from a wide age range of patients of both sexes. The newly synthesized glycosaminoglycans were identical in size and composition to those of control tissues, and their serine:sulfate ratios remained unchanged. CONCLUSION: This N-terminal peptide, which can be liberated from proteoglycan aggregates by proteolysis, potently stimulated the synthesis of proteoglycans with normal glycosaminoglycan chains. The results suggest that the N-terminal peptide may have a regulatory role in maintaining the integrity of human cartilage matrix.

Characterization of a thioredoxin-related surface protein.

A surface-associated sulphydryl (thiol) protein (SASP) constitutively present in most nucleated cells was purified from human THP-1 monocytes and rat C6 glioma cells. The human protein was similar in mass and isoelectric point and had the same N-terminal amino acid sequence to adult T-cell leukemia-derived factor (ADF), a growth factor secreted by human lymphoid cells which is able to induce increased expression of interleukin-2 receptors. A further internal amino acid sequence, determined following cleavage of human SASP with cyanogen bromide, was also identical to the corresponding sequence deduced for ADF. Samples of SASP were able to reductively depolymerize human immunoglobulin, a property shared with thioredoxin, a ubiquitous protein, almost identical to ADF, with an essential function in many thiol-dependent reducing reactions. Furthermore, SASP purified from rat C6 glioma cells had an identical N-terminal amino acid sequence to that deduced for rat liver thioredoxin, showing that they were both members of the same family of proteins. The use of membrane-impermeable thiol reagents indicated that SASP was predominantly a cell-surface protein, and was not normally secreted. This SASP protein appeared to be a surface-associated form of thioredoxin that was constitutively present in a wide range of cells and was related to ADF, a secreted form of the same protein.

Contact formation and transfer of mannose BSA gold from macrophages to cocultured fibroblasts.

When macrophages were cocultured with fibroblasts many of the cells formed firm contacts. In some of these contacts both cell types were closely apposed and in others they were more clearly separated with numerous pseudopodia extending from macrophages toward the fibroblasts. Many small vesicles similar in structure to caveoli were observed immediately beneath the plasma membrane of some fibroblasts in regions immediately adjacent to areas of contact with macrophages. The membrane integrity of both cell types was always maintained and no connecting cytoplasmic strands were observed between contacting cells. Junctions were freely permeable to ruthenium red and less permeable to the larger cationized ferritin. Gold conjugated to mannose BSA was taken up readily by macrophages but not by fibroblasts. When fibroblasts were cocultured with macrophages that had been labeled with endocytosed gold, increasing amounts were transferred to them. Gold was observed within gaps formed between cocultured cells and within recipient fibroblasts in vesicles anatomically similar to lysosomes. These points of contact thus appear to provide a series of specialized protected clefts into which directed exocytosis of ligands from donor cells can take place and from which endocytosis into recipient cells is facilitated.

Transformed SV3T3 cells have a reduced lysosomal compartment and lower levels of enzyme activity than 3T3 cells.

The secreted and intracellular activities of a number of lysosomal hydrolases were higher in 3T3 cells than in SV40-transformed cells. The number of lysosomes and their total volume were also much larger in 3T3 cells and the surface area of their lysosomal membranes was almost twice that of SV3T3 cells. These differences alone were not sufficiently large, however, to account for the disparity seen in activity of some enzymes. Gel electrophoresis showed that a number of protein components present in lysosomal membranes purified from 3T3 cells were absent from SV3T3 membrane preparations. The absence of these components may be correlated with the reduced enzyme activity of SV3T3 cells particularly with respect to beta-glucosidase and acid phosphatase, both of which are normally found associated with lysosomal membranes.

Intracellular localization of beta-glucuronidase in fibroblasts after direct transfer from macrophages.

The subcellular distribution of beta-glucuronidase acquired by deficient human fibroblasts during co-culture with peritoneal macrophages was compared with that taken up by receptor-mediated endocytosis. Labelled enzyme taken up via receptors was located initially in a low-density endosomal fraction and was transferred to lysosomes within a few minutes. The beta-glucuronidase acquired during 24 h of co-culture was present almost entirely within lysosomes and had a distribution profile identical with that of endogenous beta-hexosaminidase. Monensin prevented transfer of radiolabelled enzyme from endosomes to lysosomes and had a similar effect on the distribution of enzyme acquired by direct transfer, causing beta-glucuronidase to accumulate within endosomes. When the temperature was lowered from 37 degrees C to 19 degrees C, the rate of transfer of enzyme from endosomes to lysosomes was decreased during both direct transfer and indirect receptor-mediated endocytosis. These results show that a lysosomal enzyme acquired by direct transfer during cell-to-cell contact follows a similar intracellular route and has a similar distribution to that of enzymes taken up via cell-surface receptors.

Receptor-mediated uptake of beta-glucuronidase into primary astrocytes and C6 glioma cells from rat brain.

The ability of both primary astrocytes from rat cerebrum and a rat C6 glioma cell line to take up lysosomal enzymes by receptor-mediated endocytosis was compared. The beta-glucuronidase secreted by 3T3 fibroblasts was purified to homogeneity by antibody affinity chromatography, iodinated and used as a typical enzyme to determine the nature of receptors involved in its uptake into glial cells. Both primary astrocytes and C6 glioma cells took up 125I-labelled enzyme in a rapid and saturable manner indicative of specific receptors, while immunostaining with an anti-mouse beta-glucuronidase antibody showed that the enzyme was distributed in a mainly punctate pattern after uptake, characteristic of that of lysosomes. Subcellular fractionation of C6 glioma cells following endocytosis revealed that the enzyme became localised in lysosomes, after first passing through an endosomal compartment. Uptake of enzyme was reduced markedly after its sugar side chains had been removed with N-glycanase, indicating that endocytosis was mediated via a carbohydrate-recognising receptor. A range of carbohydrates and glycoproteins were tested for their ability to inhibit receptor-mediated endocytosis but of these only sialic acid had a notable effect. Further evidence that endocytosis of beta-glucuronidase into primary astrocytes and C6 gliomas may be mediated via sialic acid receptors was provided by the large reduction in rate of uptake observed following removal of this sugar from the enzyme with sialidase.

Cell contact and direct transfer between co-cultured macrophages and fibroblasts.

Mouse peritoneal macrophages formed attachments with beta-glucuronidase deficient human fibroblasts within an hour after co-cultures were initiated. Some of these attachments were transitory, while in others macrophages remained in firm contact with fibroblasts for many hours. Attachment of one macrophage did not prevent attachment of others, since many fibroblasts made firm contact with four or five other cells. Not all macrophages, however, attached themselves to fibroblasts. Macrophages injected with Lucifer yellow did not transfer the dye to fibroblasts with which they had made contact, nor was there any reverse transfer from injected fibroblasts to macrophages. Lucifer yellow was, however, transferred rapidly from injected fibroblasts to other adjacent fibroblasts with which they had formed gap junctions. Macrophages whose lysosomes had been pre-loaded with FITC-dextran did transfer this ligand to recipient fibroblasts, where it became localised in a perinuclear pattern with many bright punctate patches adjacent to donor macrophages. Transfer of FITC-dextran was blocked when cells were separated by nucleopore membranes in an analogous manner to transfer of endogenous lysosomal beta-glucuronidase.

Direct transfer of beta-glucuronidase from mouse macrophages to other types of cell.

Rabbit polyclonal antibodies were raised against beta-glucuronidase purified from mouse liver. This antiserum immunoprecipitated the beta-glucuronidase secreted by mouse fibroblasts but did not cross-react with the same enzyme isolated from human tissue. The beta-glucuronidase present in mouse 3T3 fibroblasts and mouse peritoneal macrophages was clearly identified by indirect immunofluorescence, using the antiserum and an FITC-conjugated second antibody, while human fibroblasts with normal levels of beta-glucuronidase activity did not fluoresce when tested with the same reagents. A range of human fibroblasts, human neuroblastoma and rat glioma cells did not fluoresce when incubated with the antibody but did fluoresce after they had been co-cultured for 24 h with mouse macrophages, showing that mouse beta-glucuronidase had been transferred from adherent macrophages into adjacent recipient cells. Transfer took place even when receptor-mediated endocytosis was blocked with a suitable competitive ligand, the transferred enzyme being visible mainly as a bright punctate fluorescence with a lysosome-like distribution. Macrophages thus have the potential to act as donors of lysosomal enzymes to a wide range of recipient cells and to transfer enzymes to them during direct cell-to-cell contact.

Fibroblasts acquire beta-glucuronidase by direct and indirect transfer during co-culture with macrophages.

Macrophages can transfer beta-glucuronidase directly to co-cultured fibroblasts during cell-to-cell contact as well as indirectly via receptor-mediated endocytosis. The degree of enzyme activity acquired by the deficient fibroblasts was determined by the ratio of donor to recipient cells and by the length of time for which cells were allowed to interact. Both mechanisms of transfer were efficient so that 70% of normal enzyme activity was restored to deficient fibroblasts after 24 h of co-culture. These observations show that macrophages have great potential as donor cells in replacement therapy for the treatment of inherited lysosomal enzyme deficiency diseases.

Iodinated fibroblast beta-glucuronidase as a ligand for receptor-mediated endocytosis.

Antibodies raised to human placental beta-glucuronidase were shown to cross-react with the beta-glucuronidase secreted by mouse 3T3 fibroblasts, but did not react with other lysosomal enzymes. The beta-glucuronidase secreted by 3T3 cells was purified 15000-fold by chromatography on an affinity column made from this antibody and resolved into a single component, of Mr 68000, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Iodinated samples of purified enzyme were taken up into mouse peritoneal macrophages by receptor-mediated endocytosis at a rate similar to that calculated previously for unlabelled enzyme, and uptake was competitively inhibited by yeast mannan. Binding of beta-glucuronidase to macrophages was saturable, with a Kd of 7 X 10(-9)l/mol, an affinity comparable with that calculated for the binding of mannosylated bovine serum albumin (Kd 1.3 X 10(-9)l/mol), a ligand specific for mannose receptors. Four times as many molecules of mannosylated albumin (12000) as of beta-glucuronidase (3000), however, bound to each cell. This purification and iodination procedure did not therefore have any adverse effect on the uptake properties of secreted beta-glucuronidase, and provides a ligand with which to investigate binding and specific endocytosis into a range of different types of cell.

Effects of mitogenic stimulation of lymphocytes on lysosomal enzyme activity.

Changes in the activities of several lysosomal enzymes were studied during transformation of mouse spleen cells in vitro. The activity of beta-glucuronidase increased during culture in the presence of T or B-cell mitogens, and lymphoblasts contained higher levels of activity than did small, non-transformed lymphocytes. Moreover, lymphoblasts in well-transformed cultures had higher activities than those in poorly-transformed cultures. The activities of other lysosomal enzymes (N-acetyl-beta-glucosaminidase, alpha-mannosidase, beta-glucosidase) also increased during mitogenic stimulation, but each at different rates, although aryl sulphatase was unaffected. Such differences may be of importance when lymphocytes are used for diagnosis of inherited lysosomal deficiency diseases.

Receptor-mediated endocytosis of fibroblast beta-glucuronidase by peritoneal macrophages.

beta-Glucuronidase secreted by mouse 3T3 fibroblasts in vitro was taken up into mouse peritoneal macrophages and into human fibroblasts by a process which was rapid and saturable. High concentrations of mannose-containing compounds inhibited uptake into macrophages but had no effect on uptake into fibroblasts. Mannose-6-phosphate inhibited uptake into both types of cell, reducing uptake into macrophages by 34% and abolishing uptake into fibroblasts completely at a concentration of 5 mM. Fructose-1-phosphate was almost equally as effective at inhibiting uptake into fibroblasts but had no effect on macrophages. Pre-treatment of beta-glucuronidase with alkaline phosphatase totally prevented its uptake into fibroblasts but had no effect on its uptake into macrophages. These results indicate that fibroblasts can secrete a lysosomal enzyme in a form recognised as a high uptake ligand not only by other fibroblasts but also by peritoneal macrophages and that endocytosis appears to be mediated by different receptors present on each type of cell. This has important implications for the potential treatment of mucopolysaccharidoses by fibroblast transplants.