Folding competence of N-terminally truncated forms of human procathepsin B

Besides acting as an inhibitor, the propeptide of human cathepsin B exerts an important auxiliary function as a chaperone in promoting correct protein folding. To explore the ability of N-terminally truncated forms of procathepsin B to fold into enzymatically active proteins, we produced procathepsin B variants progressively lacking N-terminal structural elements in baculovirus-infected insect cells. N-terminal truncation of the propeptide by up to 22 amino acids did not impair the production of activable procathepsin B. Secreted forms lacking the first 20, 21, or 22 amino acids spontaneously generated mature cathepsin B through autocatalytic processing, demonstrating that the first alpha-helix (Asp11-Arg20) is necessary for efficient inhibition of the enzyme by its propeptide. In contrast, proenzymes lacking the N-terminal part including the first beta-sheet (Trp24-Ala26) of the propeptide or containing an amino acid mutation directly preceding this beta-sheet were no longer properly folded. This shows that interactions between Trp24 of the propeptide and Tyr183, Tyr188, and Phe180 of the mature enzyme are important for stabilization and essential for procathepsin B folding. Thus, proenzyme forms missing more than the N-terminal 22 amino acids of the propeptide (notably truncated cathepsin B produced by the mRNA splice variant lacking exons 2 and 3, resulting in a propeptide shortened by 34 amino acids) are devoid of proteolytic activity because they cannot fold correctly. Thus, any pathophysiological involvement of truncated cathepsin B must be ascribed to properties other than proteolysis.     

Analysis of Fasciola cathepsin L5 by S2 subsite substitutions and determination of the P1-P4 specificity reveals an unusual preference

Fasciola parasites (liver flukes) express numerous cathepsin L proteases that are believed to be involved in important functions related to host invasion and parasite survival. These proteases are evolutionarily divided into clades that are proposed to reflect their substrate specificity, most noticeably through the S(2) subsite. Single amino acid substitutions to residues lining this site, including amino acid residue 69 (aa69; mature cathepsin L5 numbering) can have profound influences on subsite architecture and influence enzyme specificity. Variations at aa69 among known Fasciola cathepsin L proteases include leucine, tyrosine, tryptophan, phenylalanine and glycine. Other amino acids (cysteine, serine) might have been expected at this site due to codon usage as cathepsin L isoenzymes evolved, but C69 and S69 have not been observed. The introduction of L69C and L69S substitutions into FhCatL5 resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants in Fasciola. An FhCatL5 L69F variant showed an increase in the ability to cleave substrates with P(2) proline, indicating F69 variants expressed by the fluke would likely have this ability. An FhCatL2 Y69L variant showed a decreased acceptance of P(2) proline, further highlighting the importance of Y69 for FhCatL2 P(2) proline acceptance. Finally, the P(1)-P(4) specificity of Fasciola cathepsin L5 was determined and, unexpectedly, aspartic acid was shown to be well accepted at P(2,) which is unique amongst Fasciola cathepsins examined to date.     

Alternate use of divergent forms of an ancient exon in the fructose-1,6-bisphosphate aldolase gene of Drosophila melanogaster.

The fructose-1,6-bisphosphate aldolase gene of Drosophila melanogaster contains three divergent copies of an evolutionarily conserved 3' exon. Two mRNAs encoding aldolase contain three exons and differ only in the poly(A) site. The first exon is small and noncoding. The second encodes the first 332 amino acids, which form the catalytic domain, and is homologous to exons 2 through 8 of vertebrates. The third exon encodes the last 29 amino acids, thought to control substrate specificity, and is homologous to vertebrate exon 9. A third mRNA substitutes a different 3' exon (4a) for exon 3 and encodes a protein very similar to aldolase. A fourth mRNA begins at a different promoter and shares the second exon with the aldolase messages. However, two exons, 3a and 4a, together substitute for exon 3. Like exon 4a, exon 3a is homologous to terminal aldolase exons. The exon 3a-4a junction is such that exon 4a would be translated in a frame different from that which would produce a protein with similarity to aldolase. The putative proteins encoded by the third and fourth mRNAs are likely to be aldolases with altered substrate specificities, illustrating alternate use of duplicated and diverged exons as an evolutionary mechanism for adaptation of enzymatic activities.     

Exploring the role of 5' alternative splicing and of the 3'-untranslated region of cathepsin B mRNA

The cysteine peptidase cathepsin B is responsible for connective tissue breakdown in several diseases. The pathological expression of cathepsin B may depend on the structure of its mRNA. We investigated the translational efficiency of the cathepsin B mRNA untranslated regions (UTRs) using fusion constructs to green fluorescent protein (GFP) and luciferase. Transfection of fusion constructs with GFP and luciferase containing the full-length 5'-UTR, the variant lacking exon 2, and that lacking exons 2 and 3 into mammalian cells, resulted in modulation of the biosynthetic rate of cathepsin B in a cell-specific manner. Constructs missing these exons were biosynthetically more efficient than the full-length counterpart. Luciferase was cloned upstream of the 3'-UTR, downstream of the 5'-UTR, or sandwiched between the 5'- and the 3'-UTR. The UTRs of cathepsin B downregulated luciferase biosynthesis moderately when present individually, with the 3'-UTR being more efficient than the 5'-UTR, and downregulated it even more when present simultaneously. A truncated cathepsin B-GFP chimeric product derived from the 5'-UTR missing exons 2 and 3 induced cell death. The increased biosynthetic rate and abnormal trafficking of cathepsin B observed in pathologies such as cancer and osteoarthritis may depend on alternative splicing of pre-mRNA.     

The N-terminal amino acid sequences of the heavy and light chains of human cathepsin L. Relationship to a cDNA clone for a major cysteine proteinase from a mouse macrophage cell line.

Human liver cathepsin L consists of a heavy chain and a light chain with Mr values of 25,000 and 5000 respectively. The chains have been purified and their N-terminal amino acid sequences have been determined. The 40 amino acids determined from the heavy chain and 42 amino acids sequenced in the light chain are homologous with the N-terminal and C-terminal regions respectively of the superfamily of cysteine proteinases. Therefore it is likely that the two chains of cathepsin L are derived by proteolysis of a single polypeptide precursor. Of the amino acids sequenced, 81% are identical with the homologous portions of a protein sequence for a major cysteine proteinase predicted from a cDNA clone from a mouse macrophage cell line. This is the closest relative amongst the known sequences in the superfamily and strongly indicates that the protein encoded by this mRNA is cathepsin L. The mouse protein is also probably the major excreted protein of a transformed cell line [Gal & Gottesman (1986) Biochem. Biophys. Res. Commun. 139, 156-162]. The heavy chain is identical in only 71% of its residues with the sequence of ox cathepsin S, providing further evidence that this latter enzyme is probably not a species variant of cathepsin L. The relationship with a second unidentified cathepsin cDNA clone from a bovine library is much weaker (41% identity), and so this clone remains unidentified.     

The complete genomic organization of the human MUC6 and MUC2 mucin genes

The complete genomic organization of the two mucin genes MUC2 and MUC6 was obtained by comparison of new and published mRNA sequences with newly available human genomic sequence. The two genes are located 38.5 kb apart in a head-to-head orientation within a gene complex on chromosome 11p15.5. The N-terminal organization of MUC6 is highly similar to that of MUC2, containing the D1, D2, D', and D3 Von Willebrand factor domains followed by the large tandem repeat domains located in exons 31 and 30, respectively. MUC6 has a much smaller C-terminal domain (101 amino acids) encoded by 2 exons containing only the CK domain, compared with MUC2, which has a C-terminal domain of 859 amino acids containing the D4, C, D, and CK domains, encoded by 19 exons. The gene structures agreed partially but not completely with predictions from gene prediction programs.     

Transactivation of a human cytomegalovirus early promoter by gene products from the immediate-early gene IE2 and augmentation by IE1: mutational analysis of the viral proteins.

Expression from a human cytomegalovirus early promoter (E1.7) has been shown to be activated in trans by the IE2 gene products (C.-P. Chang, C. L. Malone, and M. F. Stinski, J. Virol. 63:281-290, 1989). Using wild-type and mutant viral proteins, we have defined the protein regions required for transactivation of the E1.7 promoter in IE2 and for augmentation of transactivation in the IE1 protein. Two regions of the IE2 proteins were found to be essential for transactivation. One near the amino terminus is within 52 amino acids encoded by exon 3. The second comprises the carboxyl-terminal 85 amino acids encoded by exon 5. The IE2 protein encoded by an mRNA which lacks the intron within exon 5 and the IE2 protein encoded by exon 5 had no activity for transactivation of the E1.7 promoter. Although the IE1 gene product alone had no effect on this early viral promoter, maximal early promoter activity was detected when both IE1 and IE2 gene products were present. The IE1 protein positively regulated its enhancer-containing promoter-regulatory region. The IE1 protein alone increased the steady-state level of IE2 mRNA; therefore, IE1 and IE2 are synergistic for expression from the E1.7 promoter. Like the IE2 proteins, the IE1 protein requires for activity 52 amino acids encoded by exon 3. IE1 also requires amino acids encoded by exon 4. Since the IE1 and IE2 proteins have 85 amino acids in common at the amino-terminal end encoded by exons 2 and 3, the difference between these specific transactivators resides in their carboxyl-terminal amino acids encoded by exons 4 and 5, respectively.     

Identification of interleukin-8 converting enzyme as cathepsin L

IL-8 is produced by various cells, and the NH₂-terminal amino acid sequence of IL-8 displays heterogeneity among cell types. The mature form of IL-8 has 72 amino acids (72IL-8), while a precursor form (77IL-8) of IL-8 has five additional amino acids to the 72IL-8 NH₂-terminal. However, it has been unclear how IL-8 is processed to yield the mature form. In this study, converting enzyme was purified as a single 31-kDa band on silver-stained polyacrylamide gel from 160 l of cultured fibroblast supernatant by sequential chromatography. NH₂-terminal amino acid sequence analysis revealed a sequence, EAPRSVDWRE, which was identified as a partial sequence of cathepsin L. Polyclonal antibodies raised against cathepsin L recognized the purified converting enzyme on Western blot. Moreover, human hepatic cathepsin L cleaved 77IL-8 between Arg⁵ and Ser⁶, which is the same cleavage site as the putative converting enzyme, resulting in 72IL-8 formation. These data indicate that the converting enzyme of the partially purified fraction of the human fibroblast culture supernatant was cathepsin L. Furthermore, 72IL-8 was sevenfold more potent than 77IL-8 in a neutrophil chemotaxis assay. These results show that cathepsin L is secreted from human fibroblasts in response to external stimuli and plays an important role in IL-8 processing in inflammatory sites.     

Regulation of human cathepsin B by alternative mRNA splicing: homeostasis, fatal errors and cell death

One of the control mechanisms of cathepsin B biosynthesis and trafficking operates through alternative splicing of pre-mRNA. An mRNA lacking exon 2 is more efficiently translated than that containing all exons, and may be responsible for elevated biosynthesis and enzyme routing to the extracellular space, with critical consequences for connective tissue integrity in pathologies such as cancer and arthritis. mRNA missing exons 2 and 3 encodes a truncated procathepsin B form that is targeted to mitochondria. This enzyme variant is catalytically inactive because it cannot properly fold. However, it provokes a cascade of events, which result first in morphological changes in intracellular organelles and the nucleus, finally leading to cell death.     

Cloning of cDNA for mosquito lysosomal aspartic protease. Sequence analysis of an insect lysosomal enzyme similar to cathepsins D and E.

A cDNA coding for the lysosomal aspartic protease from the mosquito (mLAP) was cloned and sequenced. The mLAP cDNA is 1420 base pairs long with an open reading frame of 387 amino acids. The deduced amino acid sequence contains a signal pre-propeptide sequence of 18 amino acids followed by 369 amino acids with a 35-amino acid putative pro-enzyme domain in the NH2-terminal. The amino acid sequence of mLAP is 92 and 81% similar to human cathepsin D and cathepsin E, respectively. Typical cleavage sites for cathepsin D processing into light and heavy chains are lacking in mLAP. A single glycosylation site occurs in the mLAP sequence at a position corresponding to the first glycosylation site of cathepsins D. The mLAP sequence shares putative phosphorylation determinants, which in cathepsins D are linked to the formation of mannose 6-phosphate. In the mosquito fat body, lysosomal enzymes specifically degrade organelles involved in the biosynthesis and secretion of vitellogenin. The mLAP mRNA accumulates to its highest level 24 h after initiation of vitellogenin synthesis and 12 h before the peak of mLAP protein accumulation and its enzymatic activity. Translational regulation of mLAP mRNA may occur. The 5'-untranslated region of mLAP mRNA is similar to elements conferring negative translational control by steroids.