Thyroglobulin type-1 domain is a structural element found in a variety of functionally unrelated proteins. It may occur singly, as in insulin-like growth factor binding proteins, or multiply up to ...eleven, as in thyroglobulin. Some of these type-1 domain containing proteins have been shown to have the ability to inhibit either cysteine or cation-dependent proteinases. For these inhibitory proteins we proposed the term thyropins. These inhibitors originate from a variety of organisms and presumably have different biological functions. One of those whose function has been studied is the p41 invariant chain fragment. In vivo it associates with the major histocompatibility complex (MHC) class II molecule. The evidence supporting its involvement as a controlling factor in the process of antigen presentation is described.
The invariant chain (Ii) is associated with major histocompatibility complex class II molecules during early stages of their intracellular transport. In an acidic endosomal/lysosomal compartment, it ...is proteolytically cleaved and removed from class II heterodimers. Participation of aspartic and cysteine proteases has been observed in in vitro degradation of Ii, but the specific enzymes responsible for its in vivo processing are as yet undefined. We have previously isolated a noncovalent complex of the lysosomal cysteine protease cathepsin L with a peptide fragment derived from the p41 form of Ii from human kidney. Here we show that this Ii fragment, which is identical to the alternatively spliced segment of p41, is a very potent competitive inhibitor of cathepsin L (equilibrium inhibition constant Ki = 1.7 X 10(-12) M). It inhibits two other cysteine proteases, cathepsin H and papain, but to much lesser extent. Cysteine proteases cathepsins B, C, and S, as well as representatives of serine, aspartic, and metalloproteases, are not inhibited at all. These findings suggest a novel role for p41 in the regulation of various proteolytic activities during antigen processing and presentation. The Ii inhibitory fragment shows no sequence homology with the known cysteine protease inhibitors, and may, therefore, represent a new class.
Summary
Activation of the CD4+ T‐cell mediated immune response relies on the proteolytic capacity of enzymes involved in modulating major histocompatibility complex (MHC) II‐associated antigen ...presentation in antigen‐presenting cells (APC). The MHC II‐associated chaperone molecule p41 isoform of invariant chain (inhibitory p41 Ii) has been suggested to regulate stability and activity of cathepsin L in these APC. In the present study the human lymph node distribution of non‐inhibitory p31 Ii and inhibitory p41 Ii have been compared by differential labelling, using two specific monoclonal antibodies. The distribution of p41 Ii, but not p31 Ii, matched the distribution of cathepsins L and H in subcapsular and cortical sinuses and germinal centres. Co‐localization of p41 Ii with cathepsin H was confirmed in strongly CD68+ sinus‐lining macrophages, acting as APC. Furthermore, p41 Ii was determined together with cathepsins L and H in tingible body macrophages, highly phagocytic, but not antigen‐presenting cells inside germinal centres. With respect to the physiological function that these two populations of macrophages have in human lymph nodes, our results support a regulatory function of p41 Ii towards cathepsins L and H in human macrophages, associated with the processes of phagocytosis rather than antigen presentation.
High levels of protease inhibitors are induced in potato leaves by wounding. These inhibitors, when ingested by Colorado potato beetle (
Leptinotarsa decemlineata Say) larvae, induce expression of ...specific proteolytic activities in the gut. Induced protease activities cannot be inhibited by potato inhibitors and thus enable the insects to overcome this defence mechanism of potato plants. The induced aminopeptidase and endoproteolytic activities both have the characteristics of cysteine proteases. Twenty-one protein inhibitors of different structural types have here been examined for their ability to inhibit these activities in vitro. Members of the cystatin superfamily were found to be poor inhibitors of the induced endoproteolytic activities, except for the third domain of human kininogen, which was a fairly strong inhibitor (75% inhibition). The strongest inhibition (85%) of induced endoproteolytic activity was obtained using structurally different thyroglobulin type-1 domain-like inhibitors—equistatin and MHC class II-associated p41 invariant fragment. Experiments performed using three synthetic substrates for endoproteases gave similar results and indicate the existence of at least three different endoproteolytic enzymes resistant to potato inhibitors. The induced aminopeptidase activity can be inhibited only by stefin family of inhibitors in cystatin superfamily. In in vivo experiments, Colorado potato beetle larvae fed on equistatin-coated potato leaves were strongly retarded in their growth and almost 50% died after 4 days. This demonstrated the potential of equistatin to protect crops from insect attack.
Thyroglobulin contains 11 repeats of a motif called thyroglobulin type-1 domain that show sequence similarity to some proteins exhibiting inhibitory activity against cysteine proteinases. Here we ...report that thyroglobulin decreases the activity of cathepsins B, H, L, and papain. To examine the possible involvement of particular type-1 domains in that decrease of activity, some individual thyroglobulin type-1 domains were expressed in E. coli. These recombinant domains proved to be substrates for cathepsins B, H, L, and papain instead of inhibitors. The cleavage points with cathepsins B and L on the second and the fourth domains were determined. The possible reasons for degradation are discussed.
A peptide fragment derived from the p41 form of the invariant chain (Ii) associated with the major histocompatibility complex (MHC) class II molecule has been shown to inhibit the mammalian lysosomal ...cysteine proteinase, cathepsin L, and to be a novel cysteine proteinase inhibitor, distinct from cystatins. Here we report that this same fragment also binds to and inhibits cruzipain, the cathepsin L-like enzyme from the protozoan parasite
Trypanosoma cruzi. The binding of the Ii fragment to cruzipain is fast (
k
ass=2.4×10
7 M
−1 s
−1) and tight (
K
i=5.8×10
−11 M). The inhibition is competitive. These results suggest the possibility of using the invariant chain as a model for the specific inhibition of cruzipain in vivo, i.e. as a potential drug to combat Chagas' disease.
Cathepsin L is a lysosomal cysteine protease involved in intracellular protein degradation. Recently, several new cysteine proteases have been identified. Human cathepsin V, a thymus- and ...testis-specific human cysteine protease, shares 78% sequence identity with human cathepsin L. Due to the strong sequence similarity, highly selective reagents are needed to elucidate the physiological functions of the two enzymes. Monoclonal antibodies (mAbs) have been prepared against recombinant human cathepsin L. Antibodies produced by five clones reacted with procathepsin L and mature cathepsin L. They also reacted with cathepsin L in complex with a peptide fragment, which is identical to the alternatively spliced segment of the p41 form of MHC Class II associated invariant chain. Two mAbs, (M105 and H102) were specific for cathepsin L, while three (N135, B145 and D24) cross-reacted with cathepsin V. None of the mAbs cross-reacted with cathepsins B, H and S. We have developed a sandwich enzyme-linked immunosorbent assay (ELISA) for quantifying cathepsin L. This sandwich ELISA uses a combination of two monoclonal antibodies which recognize different, non-overlapping epitopes on the cathepsin L molecule. The lower detection limit of the sandwich ELISA was 5 ng of cathepsin L per ml.
PDF
