Abstract
Overlapping cDNA fragments encoding avian cathepsin B were cloned from an osteoclast cDNA library and sequenced. The primary structure of the prepro enzyme deduced from this sequence has 340 amino acids. The mature portion of the enzyme is 80% identical with murine cathepsin B; regions found in other papain superfamily enzymes are conserved. In osteoclasts and cultured macrophages, which produce large quantities of cathepsin B, mRNAs of 1.8 and 2.4 kb are produced in approximately equal quantities, while cells producing smaller quantities of the enzyme produce predominantly the 2.4 kb form. This variation in mRNAs suggests transcriptional differences related to production of large quantities of the enzyme.
| Original language | English |
|---|---|
| Pages (from-to) | 69-73 |
| Number of pages | 5 |
| Journal | Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular |
| Volume | 1251 |
| Issue number | 1 |
| DOIs | |
| State | Published - Aug 16 1995 |
Keywords
- Cathepsin B
- Osteoclast
- Thiol proteinase
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In: Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular, Vol. 1251, No. 1, 16.08.1995, p. 69-73.
Research output: Contribution to journal › Article › peer-review
TY - JOUR
T1 - Avian cathepsin B cDNA
T2 - sequence and demonstration that mRNAs of two sizes are produced in cell types producing large quantities of the enzyme
AU - Dong, Sai Sai
AU - Stransky, Gregor I.
AU - Whitaker, Charles H.
AU - Jordan, Sharon E.
AU - Schlesinger, Paul H.
AU - Edwards, John C.
AU - Blair, Harry C.
N1 - Funding Information: respect to the mouse gene (glycine 203, Fig. 1). Regions commonly found in cysteine proteinases were conserved (underlined in chicken amino acid sequence in Fig. 1). Deduced structure reported here agrees with earlier reports of partial amino-acid sequence by Edman degradation, comprising = 20% of the processed enzyme \[2,19\]. Relative to the previous report of a partial cathepsin B cDNA from avian osteoclasts \[2\],t here are six differences in the deduced amino acid code of the complete cDNA (Fig. 1). These differences were investigated by direct comparisons of the sequencing gels. Three of the differences, amino acids 228-230, reflect an experimental error in the earlier publication \[2\],a frame-shift error in reading three adjacent codons, which is corrected in the current report. The other differences are single base substitutions, resulting in changes of Ala to Ser at amino acid 113, Val to Ala at 116, and Gly to Ala at 181. There are third base nucleic acid differences relative to the previous isolate at nucleotides 78 (C to T) and 264 (A to G), neither of which affects the protein. These substitutions are generally conservative, and may represent polymorphism. However, the earlier report was based on one doubly amplified PCR product, and PCR amplification errors in the earlier isolate cannot be ruled out. Comparison of the avian osteoclast enzyme to other cathepsin Bs after conservative alignment \[18\] showed percent identity relative to bovine (GenBank L06075), human (GenBank P07858), mouse (GenBank M!4222), and rat (GenBank A00977) sequences of 75, 75, 74, and 76 percent, respectively. Similarity with respect to the rat cathepsin B sequence is probably overestimated, because the rat sequence is incomplete (from amino acid 69, Fig. 1) and thus missing much of the highly variable leader sequence. These data confirm the identity of the present isolate as a cathepsin B. In keeping with divergence of the subfamilies of cysteine proteinases, identity of chicken osteoclast cathepsin B conservatively aligned \[18\]w ith other thiol proteinases was lower. Alignments with respect to full-length deduced proteins were 19% for rat cathepsin C (GenBank D90404), 19% for human cathepsin H (GenBank P09668), 15% for mouse cathepsin L (GenBank M20495), 17% for human cathepsin O (GenBank X77383), 18% for human cathepsin S (GenBank A42896), and 15% for rabbit OC-2 (GenBank D14036). Rabbit OC-2 is interesting because this enzyme, approx. 50% identical to either cathepsins L or S, was deduced from a cDNA isolated from a rabbit osteoclast library \[20\],a lthough functional studies of its role in bone degradation have not yet been reported. To determine transcript size and approximate transcript abundance in osteoclasts, macrophages, and other tissues, Northern Hybridization was performed, using a long probe from nucleotides 261 to 1023 (Fig. 1), corresponding to essentially the entire mature molecule and 12 non-coding 3' bases, extracted from the pCR II plasmid by EcoRI restriction. Results shown are from single hybridizations, but were typical of several experiments; undegraded RNA with inter-sample variation < 20% was verified by ethidium bromide staining and densitometry of 18 and 28S bands. Two mRNAs, at ~ 1.8 and 2.4 kb, at approximately equal quantities, are seen in osteoclasts at all time points (Fig. 2A). The amount of cathepsin B mRNAs seen in fresh osteoclasts varied, with individual preparations seen with both lower and higher amounts of cathepsin B mRNA. This may possibly reflect differences in activity states of the cells in the animal at time of sacrifice. There were consistent drops in both osteoclast transcripts at 1 day of isolation, with increase to baseline by 3 days, suggesting a recovery process in cells disrupted from their bone attachment. This correlates with poor bone resorption activity during this period by isolated cells \[14\]. High levels of both transcripts were consistently observed after 3-5 days in tissue culture. In isolated macrophages, cathepsin B mRNAs were not prominent, but increased with culture time with the same pattern as seen in osteoclasts after several days (Fig. 2B). The conditions used allow macrophage fusion and formation of cells resembling osteoclasts with a time-course similar to the increase in cathepsin B transcripts seen here \[21\ ]. There are smaller amounts of mRNA present in other organs tested and, in contrast to osteoclasts, the larger transcript is clearly the major product (Fig. 2C). The smaller amounts of mRNA present in these organs require long exposures for the pattern to be clearly seen (right side of Fig. 2C). We find that avian cathepsin B is quite similar in structure to murine cathepsin B, including in the size of its prepro form, key post-translational signal domains, and conservation of functional sequences. These data suggest that studies of its detailed function in avian osteoclasts are likely to reflect similar properties in mammalian cells. Further, we find that production of large quantities of avian cathepsin B by osteoclasts correlates with increased mRNA and significant amounts of a second transcript that is produced at low levels, or not at all, in other tissues. The chicken osteoclast cathepsin B transcripts reflects a second transcript is minor or non-existent in liver or other tissues, unlike the two transcripts of cathepsin B produced in murine tissues. In the mouse, transcripts at 2.2 and 4.2 kb were found in normal liver, and both were increased 2.7-3.6-fold in metastatic melanoma \[13\]. On the other hand, human tumor cells over-producing cathepsin B have been reported to show an mRNA pattern with a new transcript, similar to that seen here in normal chicken cells \[12\].T he human tumor second transcript is reported to be translated at higher relative levels than the 'housekeeping' type found in other cells \[12\]; all cells with lysosomes make small quantities of cathepsin B. Our findings therefore suggest that non-transformed cells use a similar system to increase cathepsin B production, although the mechanism is unknown. This work was supported in part by the office of Research and Development, Medical Research Service, Department of Veteran's Affairs (HCB) Funding Information: and by National Institutes of Health USA grants AR42370 (PHS, JCE) and AG12951 (HCB).
PY - 1995/8/16
Y1 - 1995/8/16
N2 - Overlapping cDNA fragments encoding avian cathepsin B were cloned from an osteoclast cDNA library and sequenced. The primary structure of the prepro enzyme deduced from this sequence has 340 amino acids. The mature portion of the enzyme is 80% identical with murine cathepsin B; regions found in other papain superfamily enzymes are conserved. In osteoclasts and cultured macrophages, which produce large quantities of cathepsin B, mRNAs of 1.8 and 2.4 kb are produced in approximately equal quantities, while cells producing smaller quantities of the enzyme produce predominantly the 2.4 kb form. This variation in mRNAs suggests transcriptional differences related to production of large quantities of the enzyme.
AB - Overlapping cDNA fragments encoding avian cathepsin B were cloned from an osteoclast cDNA library and sequenced. The primary structure of the prepro enzyme deduced from this sequence has 340 amino acids. The mature portion of the enzyme is 80% identical with murine cathepsin B; regions found in other papain superfamily enzymes are conserved. In osteoclasts and cultured macrophages, which produce large quantities of cathepsin B, mRNAs of 1.8 and 2.4 kb are produced in approximately equal quantities, while cells producing smaller quantities of the enzyme produce predominantly the 2.4 kb form. This variation in mRNAs suggests transcriptional differences related to production of large quantities of the enzyme.
KW - Cathepsin B
KW - Osteoclast
KW - Thiol proteinase
UR - http://www.scopus.com/inward/record.url?scp=0029128688&partnerID=8YFLogxK
U2 - 10.1016/0167-4838(95)00103-2
DO - 10.1016/0167-4838(95)00103-2
M3 - Article
C2 - 7647095
AN - SCOPUS:0029128688
SN - 0167-4838
VL - 1251
SP - 69
EP - 73
JO - Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
JF - Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
IS - 1
ER -