Biomedical subjects

J S Mort

Publications and source records attributed to J S Mort.

At least 19 recordsLinked to original sources

PubMed2006

The cleavage of biglycan by aggrecanases.

OBJECTIVE: Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4] and aggrecanase-2 (ADAMTS-5) have been named for their ability to degrade the proteoglycan aggrecan. While this may be the preferred substrate for these enzymes, they are also able to degrade other proteins. The aim of this work was to determine whether the aggrecanases could degrade biglycan and decorin. METHODS: Biglycan, decorin and aggrecan were purified from human and bovine cartilage and subjected to degradation by recombinant aggrecanase-1 or aggrecanase-2. In vitro degradation was assessed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and immunoblotting, and the cleavage site in biglycan was determined by N-terminal amino acid sequencing. SDS/PAGE and immunoblotting were also used to assess in situ degradation in both normal and arthritic human articular cartilage. RESULTS: Both aggrecanase-1 and aggrecanase-2 are able to cleave bovine and human biglycan at a site within their central leucine-rich repeat regions. Cleavage occurs at an asparagine-cysteine bond within the fifth leucine-rich repeat. In contrast, the closely related proteoglycan decorin is not a substrate for the aggrecanases. Analysis of human articular cartilage from osteoarthritic (OA) and rheumatoid arthritic (RA) joints showed that a biglycan degradation product of equivalent size is present in the extracellular matrix. No equivalent degradation product was, however, detectable in normal adult human articular cartilage. CONCLUSION: Biglycan, which is structurally unrelated to aggrecan, can act as a substrate for aggrecanase-1 and aggrecanase-2, and these proteinases may account for at least part of the biglycan degradation that is present in arthritic cartilage.

ADAM Proteins↗

PubMed2006

Defects in articular cartilage metabolism and early arthritis in fibroblast growth factor receptor 3 deficient mice.

Fibroblast growth factor (FGF) receptor 3 has been identified as a key regulator of endochondral bone development and of post-natal bone metabolism through its action on growth plate chondrocytes and osteoblasts, respectively. It has also been shown to promote chondrogenesis and cartilage production by cultured pre-chondrogenic cells in response to FGF18. In the current studies, we show that the absence of signaling through Fgfr3 in the joints of Fgfr3(-/-) mice leads to premature cartilage degeneration and early arthritis. Degenerative changes in cartilage matrix included excessive proteolysis of aggrecan core protein and type II collagen, as measured by neo-epitope immunoreactivity. These changes were accompanied by increased expression of metalloproteinase MMP13, type X collagen, cellular hypertrophy and loss of proteoglycan at the articular surface. Using a novel micro-mechanical indentation protocol, it was shown that articular cartilage in the humeral head of 4-month-old Fgfr3(-/-) mice was less resistant to compressive force and less stiff than that of littermate controls. These results identify Fgfr3 signaling as a potential target for intervention in degenerative disorders of cartilage metabolism.

Aggrecans↗

PubMed2001

Soluble recombinant neprilysin induces aggrecanase-mediated cleavage of aggrecan in cartilage explant cultures.

Neprilysin (neutral endopeptidase, enkephalinase, CALLA, CD10, NEP) is a regulatory Zn metallopeptidase expressed in the brush border membranes of the kidney and has been found in porcine chondrocytes and rat articular cartilage as well as other cell types and tissues. Although its function in cartilage is not currently known, previous observations of high levels of NEP enzymatic activity in the synovial fluid of arthritic patients and on the chondrocyte membranes of human osteoarthritic cartilage have led to the hypothesis that NEP is involved in the inflammation or degradation pathways in articular cartilage. Our study localized endogenous NEP to the membranes of mature bovine articular chondrocytes in a tissue explant model and demonstrated that the addition of soluble recombinant NEP (sNEP) to the culture medium of bovine cartilage explants leads to the degradation of aggrecan through the action of aggrecanase. A 6-day exposure to sNEP was necessary to initiate the degradation, suggesting that the chondrocytes were responding in a delayed manner to an altered composition of regulatory peptides. This NEP-induced degradation was completely inhibited by the NEP inhibitors thiorphan and phosphoramidon. These results suggest that NEP is present as a transmembrane enzyme on articular chondrocytes where it can cleave regulatory peptides and lead to the induction of aggrecanase.

Aggrecans↗

PubMed2001

Pathways by which interleukin 17 induces articular cartilage breakdown in vitro and in vivo.

Overexpression of interleukin (IL-)17 has recently been shown to be associated with a number of pathological conditions. Because IL-17 is found at high levels in the synovial fluid surrounding cartilage in patients with inflammatory arthritis, the present study determined the direct effect of IL-17 on articular cartilage. As shown herein, IL-17 was a direct and potent inducer of matrix breakdown and an inhibitor of matrix synthesis in articular cartilage explants. These effects were mediated in part by leukemia inhibitory factor (LIF), but did not depend on interleukin-1 activity. The mechanism whereby IL-17 induced matrix breakdown in cartilage tissue appeared to be due to stimulation of activity of aggrecanase(s), not matrix metalloproteinase(s). However, IL-17 upregulated expression of matrix metalloproteinase(s) in chondrocytes cultured in monolayer. In vivo, IL-17 induced a phenotype similar to inflammatory arthritis when injected into the intra-articular space of mouse knee joints. Furthermore, a related protein, IL-17E, was found to have catabolic activity on human articular cartilage. This study characterizes the mechanism whereby IL-17 acts directly on cartilage matrix turnover. Such findings have important implications for the treatment of degenerative joint diseases such as arthritis.

Animals↗

PubMed2001

Articular cartilage and changes in arthritis: matrix degradation.

While many proteases in articular cartilage have been described, current studies indicate that members of two families of metalloproteases - MMPs and the ADAMTSs - are responsible for the degradation of the major components of this tissue. Collagenases (MMPs) make the first cleavage in triple-helical collagen, allowing its further degradation by other proteases. Aggrecanases (ADAMTSs), in conjunction with other MMPs, degrade aggrecan, a component of the proteoglycan aggregate. Antineoepitope antibodies that recognize the cleavage products of collagen and aggrecan generated by these enzymes are now available and are being used to detect the sites of action and to quantitate degradation products.

Arthritis↗

PubMed2001

Enzymes active in the areas undergoing cartilage resorption during the development of the secondary ossification center in the tibiae of rats aged 0-21 days: II. Two proteinases, gelatinase B and collagenase-3, are implicated in the lysis of collagen fibrils.

In the transformation of the cartilaginous epiphysis into bone, the first indication of change in the surfaces destined for resorption is the cleavage of aggrecan core protein by unidentified matrix metalloproteinases (MMPs) (Lee et al., this issue). In cartilage areas undergoing resorption, the cleavage leaves as superficial, 6-microm-thick band of matrix, referred to as "pre-resorptive layer." This layer harbors G1-fragments of the aggrecan core protein within a framework of collagen-rich fibrils exhibiting various stages of degeneration. Investigation of this layer in every resorption area by gelatin histozymography and TIMP-2 histochemistry demonstrates the presence of an MMP whose histozymographic activity is inhibited by such a low dose of the inhibitor CT1746 as to identify it as gelatinase A or B. Attempts at blocking the histozymographic reactions with neutralizing antibodies capable of inhibiting either gelatinase A or B reveals that only those against gelatinase B do so. Immunostaining of sections with anti-gelatinase B IgG confirms the presence of gelatinase B in every pre-resorptive layer, that is, at the blind end of excavated canals (stage I; 6-day-old rats), at sites along the walls of the forming marrow space (stage II; 7days), at sites within the walls of this space as it becomes the ossification center (stage III; 9 days) and along the wall of the maturing center (stage IV; 10-21 days). We also report the presence of collagenase-3 in precisely the same sites, possibly as active enzyme, but this remains to be proven. Because the results reveal that collagenase-3 is present beside gelatinase B in every pre-resorptive layer and, because these sites exhibit various signs of degradation including fibrillar debris, reduction in fibril number, or overt loss, we propose that gelatinase B and collagenase-3 mediate the lysis of this pre-resorptive layer-most likely through a cooperative attack leading to the disintegration of the collagen fibril framework.

Animals↗

PubMed2001

Enzymes active in the areas undergoing cartilage resorption during the development of the secondary ossification center in the tibiae of rats ages 0-21 days: I. Two groups of proteinases cleave the core protein of aggrecan.

The formation of a secondary ossification center in the cartilaginous epiphysis of long bones requires the excavation of canals and marrow space and, therefore, the resorption of cartilage. On the assumption that its resorption requires the lysis of the major cartilage component aggrecan, it was noted that the core protein may be cleaved in vitro by proteinases from two subfamilies: matrix metalloproteinases (MMPs) and aggrecanases. Such cleavage results in aggrecan being replaced by a fragment of itself referred to as a "G1-fragment." To find out if this cleavage occurs in the developing epiphysis of the rat tibia, the approach has been to localize the G1 fragments. For this purpose two neoepitope antisera were applied, one capable of recognizing the MMP-generated G1-fragment that bears the C-terminus ...FVDIPEN341 and the other capable of recognizing the aggrecanase-generated G1-fragment that carries the C-terminus ...NITEGE373. With the aid of these antisera, we report here that aggrecan cleavage is localized to newly developed sites of erosion. Thus, at 6 days of age, canals allowing the entry of capillaries are dug out from the surface of the epiphysis in a radial direction (stage I), whereas immunostaining indicative of aggrecan cleavage by MMPs appears at the blind end of each canal. The next day, the canal blind ends fuse to create a marrow space in the epiphysis (stage II), whereas immunostaining produced by MMPs occurs along the walls of this space. By 9 days, clusters of hypertrophic chondrocytes are scattered along the marrow space wall to initiate the formation of the secondary ossification center (stage III), where the resorption sites are unreactive to either antiserum. From the 9th to the 21st day, the center keeps on enlarging and, as the distal wall of the marrow space recedes, it is intensely immunostained with both antisera indicating that both MMPs and aggrecanases are involved in this resorption. We conclude, that both enzyme subfamilies contribute to the lysis of aggrecan. However, the results suggest that the respective subfamilies target different sites and even stages of development in the tissue, suggesting some diversity in the mode of aggrecan lysis during the excavation of a secondary ossification center.

Aggrecans↗

PubMed2000

Cystatin inhibition of cathepsin B requires dislocation of the proteinase occluding loop. Demonstration By release of loop anchoring through mutation of his110.

Cystatins A and C were both shown to inhibit cathepsin B by a two-step mechanism, involving an initial weak interaction followed by a conformational change. Disruption of the major salt bridge anchoring the occluding loop of cathepsin B to the main body of the enzyme by mutation of His110 to Ala converted the binding to an apparent one-step reaction. The second step of cystatin binding to cathepsin B must therefore be due to the inhibitor having to alter the conformation of the enzyme by displacing the occluding loop to allow a tight complex to be formed. Cystatin A was appreciably less effective in displacing the loop than cystatin C, resulting in a considerably lower overall inhibition rate constant.

Alanine↗

PubMed2000

The slow-binding inhibition of cathepsin K by its propeptide.

A peptide corresponding to the full-length proregion (amino acids 16-114) of human cathepsin K was expressed and purified from Escherichia coli. This recombinant propeptide was investigated for its ability to inhibit the activity of three cysteine proteinases: cathepsins K, L, and B. Kinetic studies showed the propeptide to be a potent slow-binding inhibitor of its parent enzyme with a K(i) = 2. 61 nM at pH 6. This inhibition was pH-dependent, with a decrease in pH from 6 to 4 leading to a concomitant increase in K(i) to 147 nM. The propeptide also inhibited cathepsin L with a K(i) = 26.1 nM at pH 6, but showed little inhibition of cathepsin B at concentrations up to 400 nM.

Antibodies, Monoclonal↗

PubMed2000

Cathepsin-B fusion proteins misroute secretory protein partners such as the proprotein convertase PC2-7B2 complex toward the lysosomal degradation pathways.

A general strategy is presented for the dominant negative reduction in the levels of heterodimeric soluble proteins within the secretory pathway through fusion of one of its partners C-terminal to the lysosomal enzyme cathepsin B (CB). Stable transfectants of CB-7B2 chimeras in AT20 cells result in a drastic reduction of the endogenous levels of its partner, the proprotein convertase PC2. This dominant negative suppressive effect requires active CB. It was partially reversed by NH(4)Cl, the cell-permeable CB inhibitor CA-074Me, but not by the proteasome inhibitor Lactacystin, suggesting the potential participation of the lysosomal/endosomal degradative pathway in this process.

Ammonium Chloride↗

PubMed2000

Optimization of detergents for the assay of cathepsins B, L, S, and K.

The differential effects of representative, commonly available ionic (SDS), nonionic (Brij 35, Tween 20, and Triton X-100), and zwitterionic (Chaps) detergents on the catalytic activity and properties of human cathepsins B, L, S, and K were examined. The presence of detergents in the assay buffer affected the activity of cathepsins to variable extents; Chaps enhanced the activity of all the enzymes while SDS was most detrimental. Tween 20 lowered cathepsin S activity, while it slightly enhanced that of all other cathepsins studied. The presence of detergents in the activation buffer was clearly beneficial to both cathepsins L and K, possibly by favoring the release of the enzyme from the walls of the incubation vessel. Overall, the results indicate that Chaps is the optimal detergent for use with this family of enzymes.

Cathepsin B↗

PubMed2000

Proteolysis of human bone collagen by cathepsin K: characterization of the cleavage sites generating by cross-linked N-telopeptide neoepitope.

An immunoassay for cross-linked N-telopeptides of type I collagen (NTx) in urine or serum has proven to give a sensitive index of osteoclast-mediated bone resorption. We show that recombinant human cathepsin K is highly active in releasing the NTx neoepitope in 100% yield from bone type I collagen. Cathepsins S, L, and B were also active but at 57%, 36%, and 27% of the yield of K, respectively. The matrix metalloproteinases that were tested, stromelysin, collagenase 3, or matrilysin, did not produce any immunoreactivity. Cathepsin K also acted on demineralized bone matrix, releasing NTx epitope and completely dissolving the bone particles in 24-48 h. Proteolytic cleavage of a G-L peptide bond in the alpha2(I)N-telopeptide was shown to be required for recognition by monoclonal antibody 1H11. Peptide analysis identified bonds in the N-telopeptide and helical cross-linking domains adjacent to the cross-linking residues at which cathepsin K cleaved in bone collagen. The sites were consistent with the known substrate specificity of cathepsin K, which prefers a hydrophobic residue or proline in the critical P2 position. The NTx peptides generated by cathepsin K were of low molecular weight, in the range previously found in human urine. Because cathepsin K appears to be essential for the normal resorption of mineralized bone matrix by osteoclasts, these findings help explain the specificity and responsiveness of NTx as a marker of osteoclastic bone resorption in vivo.

Adult↗

PubMed1999

Negative regulation of epidermal growth factor signaling by selective proteolytic mechanisms in the endosome mediated by cathepsin B.

We have investigated the relevant protease activity in rat liver, which is responsible for most of the receptor-mediated epidermal growth factor (EGF) degradation in vivo. EGF was sequentially cleaved by endosomal proteases at a limited number of sites, which were identified by high performance liquid chromatography and mass spectrometry. EGF proteolysis is initiated by hydrolysis at the C-terminal Glu(51)-Leu(52) bond. Three additional minor cleavage sites were identified at positions Arg(48)-Trp(49), Trp(49)-Trp(50), and Trp(50)-Glu(51) after prolonged incubation. Using nondenaturating immunoprecipitation and cross-linking procedures, the major proteolytic activity was identified as that of the cysteine protease cathepsin-B. The effect of injected EGF on subsequent endosomal EGF receptor (EGFR) proteolysis was further evaluated by immunoblotting. Using endosomal fractions prepared from EGF-injected rats and incubated in vitro, the EGFR was lost with a time course superimposable with the loss of phosphotyrosine content. The cathepsin-B proinhibitor CA074-Me inhibited both in vivo and in vitro the endosomal degradation of the EGFR and increased the tyrosine phosphorylation states of the EGFR protein and the molecule SHC within endosomes. The data, therefore, describe a unique pathway for the endosomal processing of internalized EGF receptor complexes, which involves the sequential function of cathepsin-B through selective degradation of both the ligand and receptor.

Amino Acid Sequence↗

PubMed1999

Aggrecanase versus matrix metalloproteinases in the catabolism of the interglobular domain of aggrecan in vitro.

The importance of aggrecanase versus matrix metalloproteinase (MMP) enzymic activities in the degradation of aggrecan in normal and osteoarthritic (OA) articular cartilage in vitro was studied in order to further our understanding of the potential role of these two enzyme activities in aggrecan catabolism during the pathogenesis of cartilage degeneration. Porcine and bovine articular cartilage was maintained in explant culture for up to 20 days in the presence or absence of the catabolic stimuli retinoic acid, interleukin-1 or tumour necrosis factor-alpha. Release of proteoglycan from cartilage was measured as glycosaminoglycan (GAG) release using a colorimetric assay. Analysis of proteoglycan degradation products, both released into culture media and retained within the cartilage matrix, was performed by Western blotting using antibodies specific for the N- and C-terminal neoepitopes generated by aggrecanase- and MMP-related catabolism of the interglobular domain of the aggrecan core protein (IGD). In addition, studies determining the mRNA expression for MMP-3 and MMP-13 in these same cultures were undertaken. These analyses indicated that all three catabolic agents stimulated the release of >80% of the GAG from the articular cartilage over 4 days. The degree of GAG release corresponded to an increase in aggrecanase-generated aggrecan catabolites released into the media and retained within the cartilage. Importantly, there was no evidence for the release of MMP-generated aggrecan metabolites into the medium, nor the accumulation of MMP-generated catabolites within the tissue in these same cultures. Expression of the mRNAs for two MMPs known to be capable of degrading the aggrecan IGD, MMP-3 and MMP-13, was detected. However, increased expression of these MMPs was not correlated with aggrecan degradation. Analyses using porcine cartilage, cultured with or without catabolic stimulation for 12 h to 20 days, indicated that primary cleavage of the IGD by aggrecanase was responsible for release of aggrecan metabolites at both the early and late time points of culture. Cultures of late-stage OA human articular cartilage samples indicated that aggrecanase activity was upregulated in the absence of catabolic stimulation when compared with normal porcine or bovine cartilage. In addition, even in this late-stage degenerate cartilage, aggrecanase and not MMP activity was responsible for the release of the majority of aggrecan from the cartilage. This study demonstrates that the release of aggrecan from both normal and OA cartilage in response to catabolic stimulation in vitro involves a primary cleavage by aggrecanase and not MMPs.

Aggrecans↗

PubMed1999

Age-related changes in fibromodulin and lumican in human intervertebral discs.

STUDY DESIGN: An analysis of proteoglycans of the intervertebral disc using immunoblotting of tissue extracts. OBJECTIVES: To investigate the changes in structure and abundance of fibromodulin and lumican in human intervertebral discs during aging and degeneration. SUMMARY OF BACKGROUND DATA: Fibromodulin and lumican are keratan sulfate proteoglycan constituents of the disc's extracellular matrix, whose interaction with collagen fibrils may contribute to the mechanical properties of the tissue. Changes in their abundance and/or structure that occur with aging and degeneration therefore may have an impact on disc function. METHODS: Lumbar intervertebral discs were obtained from individuals of different ages, and extracts of anulus fibrosus and nucleus pulposus were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using antibodies specific for fibromodulin and lumican. RESULTS: The major changes in abundance observed with age were a decrease in fibromodulin in the adult nucleus pulposus and an increase in lumican in anulus fibrosus during early juvenile development. In addition, fibromodulin in the anulus fibrosus exhibited a structural change with increasing age, characterized by a shift toward the predominance of its glycoprotein form lacking keratan sulfate. Fibromodulin was more abundant in the anulus fibrosus than in nucleus pulposus at all ages, whereas lumican was much more abundant in nucleus pulposus than in anulus fibrosus in the young juvenile; in the adult, however, lumican was present in comparable levels in both tissues. With increasing degrees of degeneration, fibromodulin exhibited an increase in abundance. CONCLUSIONS: Growth, aging, and degeneration of the intervertebral disc are associated with changes in the abundance and structure of fibromodulin and lumican, which presumably influence the functional properties of the tissue.

Adolescent↗

PubMed1999

Resistance of small leucine-rich repeat proteoglycans to proteolytic degradation during interleukin-1-stimulated cartilage catabolism.

A bovine nasal-cartilage culture system has been utilized to analyse the catabolic events occurring in response to interleukin-1beta over a 14-day period. An early event following the start of interleukin-1 treatment was the release of glycosaminoglycan into the culture medium. This release was accompanied by the appearance in the tissue, and shortly thereafter also in the culture media, of a globular domain (G1)-containing aggrecan degradation product generated by the action of aggrecanase. Link protein was also released from the cartilage with a similar timeframe to that of the G1 fragment, although there was no evidence of its proteolytic degradation. By comparison with aggrecan, the small leucine-rich repeat proteoglycans decorin, biglycan and lumican showed a resistance to both proteolytic cleavage and release throughout the culture period. In contrast, fibromodulin exhibited a marked decrease in size after day 4, presumably due to proteolytic modification, but the major degradation product was retained throughout the culture period. Also in contrast with the early changes in the components of the proteoglycan aggregate, type II collagen did not display signs of extensive degradation until much later in the culture period. Collagen degradation products compatible with collagenase action first appeared in the medium by day 10 and increased thereafter. These data demonstrate that the leucine-rich repeat proteoglycans are resistant to proteolytic action during interleukin-1-stimulated cartilage catabolism, compared with aggrecan. This resistance and continued interaction with the surface of the collagen fibrils may help to stabilize the collagen fibrillar network and protect it from extensive proteolytic attack during the early phases of cartilage degeneration.

Aggrecans↗

PubMed1999

The occluding loop in cathepsin B defines the pH dependence of inhibition by its propeptide.

Papain-like proenzymes are prone to autoprocess under acidic pH conditions. Similarly, peptides derived from the proregion of cathepsin B are potent pH-dependent inhibitors of that enzyme; i.e., at pH 6.0 the inhibition of human cathepsin B by its propeptide is defined by slow binding kinetics with a Ki of 3.7 nM and at pH 4.0 by classical kinetics with a Ki of 82 nM. This pH dependency is essentially eliminated either by the removal of a portion of the enzyme's occluding loop through deletion mutagenesis or by the mutation of either residue Asp22 or His110 to alanine; e.g., the mutant enzyme His110Ala is inhibited by its propeptide with Ki's of 2.0 +/- 0.3 nM at pH 4.0 and 1.1 +/- 0.2 nM at pH 6.0. For the His110Ala mutant the inhibition also displays slow binding kinetics at both pH 4.0 and pH 6.0. As shown by the crystal structure of mature cathepsin B [Musil, D., et al. (1991) EMBO J. 10, 2321-2330] Asp22 and His110 form a salt bridge in the mature enzyme, and it has been shown that this bridge stabilizes the occluding loop in its closed position [Nägler, D. K., et al. (1997) Biochemistry 36, 12608-12615]. Thus the pH dependency of propeptide binding can be explained on the basis of a competitive binding between the occluding loop and the propeptide. At low pH, when the Asp22-His110 pair forms a salt bridge stabilizing the occluding loop in its closed conformation, the loop more effectively competes with the propeptide than at higher pH where deprotonation of His110 and the concomitant destruction of the Asp22-His110 salt bridge results in a destabilization of the closed form of the loop. The rate of autocatalytic processing of procathepsin B to cathepsin B correlates with the affinity of the enzyme for its propeptide rather than with its catalytic activity, thus suggesting a possible influence of occluding loop stability on the rate of processing.

Amino Acid Sequence↗

PubMed1999

Interdependency of sequence and positional specificities for cysteine proteases of the papain family.

The specificity of cysteine proteases is characterized by the nature of the amino acid sequence recognized by the enzymes (sequence specificity) as well as by the position of the scissile peptide bond (positional specificity, i.e., endopeptidase, aminopeptidase, or carboxypeptidase). In this paper, the interdependency of sequence and positional specificities for selected members of this class of enzymes has been investigated using fluorogenic substrates where both the position of the cleavable peptide bond and the nature of the sequence of residues in P2-P1 are varied. The results show that cathepsins K and L and papain, typically considered to act strictly as endopeptidases, can also display dipeptidyl carboxypeptidase activity against the substrate Abz-FRF(4NO2)A and dipeptidyl aminopeptidase activity against FR-MCA. In some cases the activity is even equal to or greater than that observed with cathepsin B and DPP-I (dipeptidyl peptidase I), which have been characterized previously as exopeptidases. In contrast, the exopeptidase activities of cathepsins K and L and papain are extremely low when the P2-P1 residues are A-A, indicating that, as observed for the normal endopeptidase activity, the exopeptidase activities rely heavily on interactions in subsite S2 (and possibly S1). However, cathepsin B and DPP-I are able to hydrolyze substrates through the exopeptidase route even in absence of preferred interactions in subsites S2 and S1. This is attributed to the presence in cathepsin B and DPP-I of specific structural elements which serve as an anchor for the C- or N-terminus of a substrate, thereby allowing favorable enzyme-substrate interaction independently of the P2-P1 sequence. As a consequence, the nature of the residue at position P2 of a substrate, which is usually the main factor determining the specificity for cysteine proteases of the papain family, does not have the same contribution for the exopeptidase activities of cathepsin B and DPP-I.

Animals↗