Major histocompatibility complex class II-associated p41 invariant chain fragment is a strong inhibitor of lysosomal cathepsin L.

Abstract

SummaryThe 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 K~ = 1.7 • 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 a11. 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. MHC class II molecules are transmembrane glycopro-.teins, composed of polymorphic ~x and [3 chains, whose crucial function is to present bound antigenic peptides to CD4 + T lymphocytes (1). Shortly after their synthesis in the endoplasmic reticulum (ER) 1, class II dimers associate with a nonpolymorphic type II transmembrane protein called the invariant chain (Ii) (2). The resulting class II-Ii complexes exit the ER, and are transported through the Golgi apparatus to the endocytic pathway (3, 4).Ii contributes in a number of ways to the proper functioning of MHC class II molecules. These include promoting effective association and folding of newly synthesized oL and [3 subunits (5), increasing transit of assembled heterodimers out of the ER (6), blocking of peptide binding 1Abbreviations used in this paper: AS, additional segment of p41; CPI, cysteine protease inhibitor; DTE, dithioerithritol; Elk, endoplasmic reticulure; li, invariant chain; k, pseudo first order rate constant; k~, second order rate constant for complex formation; kai~, dissociation rate constant; Ki, equilibrium inhibition constant; Kin, Michaelis constant; -MCA, 4-methyl-7-coumarylamide; vs, steady-state velocity; vz, initial velocity; Z-, benzyloxycarbonyl. This paper is dedicated to Professor Hans Fritz on the occasion of his 60th birthday.to class II molecules in the early compartments of the biosynthefc pathway (7), and sorting class II molecules into appropriate endocytic organelles (8, 9).After arrival in endosomes, the COOH-terminal lumenal domain of Ii is degraded in distinct steps from the COOH terminus of the molecule, resulting in the sequential formation of processing intermediates, each lacking increasin...

Diverse regulatory roles for lysosomal proteases in the immune response

“…The p41 isoform of the Ii, which is abundantly expressed in some DC, turned out to be a potent inhibitor of cathepsin L [20] and recently was shown to inhibit other cathepsins [21]. However, the net effect of p41 on cathepsin L activity remains unresolved since it also acts as a cathepsin L chaperone and increases active cathepsin L protein levels in bone marrow macrophages [22].…”

“…Other recent studies report a transient arrest of DC migration following TLR signalling that correlated with loss of actin-rich matrix metalloproteaseorganising podosomes, which may also be required for DC exit from tissues [19]. Taken together, these studies support the idea that DC exit from tissues is coupled to completion of the earlier stages of antigen capture and Ii processing.The p41 isoform of the Ii, which is abundantly expressed in some DC, turned out to be a potent inhibitor of cathepsin L [20] and recently was shown to inhibit other cathepsins [21]. However, the net effect of p41 on cathepsin L activity remains unresolved since it also acts as a cathepsin L chaperone and increases active cathepsin L protein levels in bone marrow macrophages [22].…”

MHC Class II Antigen Presentation by Dendritic Cells Regulated through Endosomal Sorting

“…Because of alternative splicing, mice and humans also express p41 and p43, respectively Koch et al 1987;O'Sullivan et al 1987). The additional sequence in their exoplasmic domain has protease inhibitory ac-tivity especially to the cysteine protease cathepsin L (Koch et al 1989;Bevec et al 1996;Mihelič et al 2008) and influences the antigen-processing capacity of endosomes (Peterson and Miller 1992). In humans, but not in mice, an alternative start codon provides two additional Ii isoforms, p35 and p45, carrying a cytoplasmic extension that is shielded only after correct folding of the MHCII -Ii complex and, when exposed, serves as an ER-retention motif.…”

A fragment of the major histocompatibility complex class II – associated p41 invariant chain inhibits cruzipain, the major cysteine proteinase from Trypanosoma cruzi

“…The concentration of active cruzipain was determined using the cysteine proteinase inhibitor Ep-475, and the Ii fragment was titrated with active-site-titrated cathepsin L as previously described [15].…”

“…Recently, a novel property of Ii has been established. A 65 amino acid fragment from Ii exhibits strong inhibitory activity against cathepsin L (Äj = 1.7 pM), and also inhibits moderately cathepsin H (^ = 5.3 nM) and papain (^i=1.4 nM) [15]. It corresponds to the alternatively spliced fragment, which is present only in the minor, p41, but not in the major, p31, form of Ii.…”