Extracellular-matrix degradation at acid pH. Avian osteoclast acid collagenase isolation and characterization.
Osteoclasts degrade bone matrix, which is mainly type I collagen and hydroxyapatite, in an acidic extracellular compartment. Thus we reasoned that osteoclasts must produce an acid collagenase. We purified this enzyme, a 31 kDa protein, from avian osteoclast lysates (in 100 mM acetate/1 mM CHAPS/1 mM dithiothreitol, pH 4.4), fractionated by (NH2)2SO4 precipitation, gelatin-affinity, cation exchange, and gel filtration. Fraction activity was measured using diazotized collagen or 3H-labelled cross-linked collagen (decalcified and trypsin-treated metabolically L-[4,5-3H]proline-labelled bone) as substrates. Iodoacetate, leupeptin, antipain, pepstatin and mercurials inhibited collagenolysis by the isolated proteinase; mercurial derivatives could not be re-activated by dithiothreitol. Collagen degradation was maximal at pH 4.4; purified proteinase reproduced the collagenolytic activity of cell lysates. The N-terminal amino acid sequence from the isolated protein and its CNBr degradation fragments showed sequence similarity to mammalian cathepsin Bs, and near-identity with avian liver cathepsin B. Peptide substrate specificity of the osteoclastic enzyme resembled those of mammalian cathepsin B and its avian liver counterpart, but degradation of low-molecular-mass substrates by the osteoclastic enzyme was slower, reflecting generally lower kcat. values. Further, kcat/Km varied less between arginine-containing substrates than for previously reported cathepsin Bs, indicating different substrate specificity of the osteoclast enzyme. Polyclonal antibody raised to a 25 kDa fragment of the enzyme recognized a single 31 kDa band in SDS/PAGE of osteoclast lysates blotted to poly(vinylidene difluoride), adsorbed collagenolytic activity of osteoclast lysates, and stained avian osteoclasts in tissue sections. Degenerate sense- and antisense-oligonucleotide primers, predicted from segments of primary amino acid sequence, amplified a 486 bp DNA fragment; this was cloned and sequenced. Of 162 amino acids encoded, 77% are identical with those of human cathepsin B; hybridization identified a 2.4 kb RNA in osteoclast lysates. We conclude that the major avian osteoclast collagenolytic enzyme is a cathepsin B, whose activity varies from other enzymes of its class.
Blair
H C
HC
Department of Veterans' Affairs Medical Center, Birmingham, AL 35294.
Teitelbaum
S L
SL
Grosso
L E
LE
Lacey
D L
DL
Tan
H L
HL
McCourt
D W
DW
Jeffrey
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001857:Bone Matrix / Q000378:metabolism; D002401:Cathepsin B / Q000737:chemistry / Q000235:genetics; D002645:Chickens; D002845:Chromatography; D017364:Collagenases / Q000302:isolation & purification* / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D004229:Dithiothreitol / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism*; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010010:Osteoclasts / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D010446:Peptide Fragments; D004247:DNA; D002401:Cathepsin B; D017364:Collagenases; D004229:Dithiothreitol