Sequencing and Characterization of the Soybean Leaf Metalloproteinase

Abstract

A novel zinc endoproteinase has been sequenced and characterized from soybean leaves (Glycine max var Williams 82) and has been designated as Protein Identification Resource accession No. A41820 SMEP1 (soybean metalloendoproteinase 1). Comparison of the primary amino acid sequence with other zinc proteinases revealed the enzyme to be a new member of the matrix metalloproteinase (MMP) family of enzymes. SMEP was found to have MMP cleavage specificity toward peptide substrates and the enzyme is specifically inhibited by naturally occurring tissue inhibitors of MMPs through a high-affinity interaction (inhibitor concentration resulting in an approximate 50% decrease in enzyme activity = 23 x 10-molar). of SMEP1 and further characterization of the plant protein that places the enzyme within the MMP family. MATERIALS AND METHODSChemical Sequencing SMEP1 was purified for sequencing as previously described (10) followed by HPLC purification in a Polypore RP column (2.1 x 30 mm, Brownlee) using a linear gradient of acetonitrile/0.1% TFA (8-80%) over 30 min. Reduction and alkylation were performed by incubating the protein in 6 M guanidine-HCL, 1 M Tris, 20 mm DTT (Calbiochem), 10 mm EDTA at pH 8.6 for 1 h at 370C under nitrogen followed by addition of 4-vinylpyridine (Sigma) to 50 mi. All sequence-grade proteinase (Boehinger Mannheim, Indianapolis, IN) digestions, except pepsin (Pierce Chemical Co., Rockford, IL), were performed at room temperature over 16 to 19 h under conditions recommended by the suppliers and as previously described (9). For Asp-N digests, the protein was pretreated by incubation with 4 M urea for 30 min at 500C. Digestion with immobilized pepsin was performed in 0.1S% formic acid with the addition of a few beads for 15 min. Reduction of methionine sulfoxide was performed using 750 mm DTT for 48 h at 370C. Automated Edman degradations were performed using the ABI 477A liquid pulse sequencer and an ABI 120A PTH analyzer. Mass spectra of the intact SMEP1 protein or peptide fragments were collected on a Sciex API-III mass spectrometer equipped with ion-spray inlet. Spectra were scanned from m/z 300 to m/z 2200 repetitively at 5 s per scan and several spectra were averaged for molecular mass calculations. Cleavage SpecificityDigests were conducted with 100 /LM substrate and 20 nm enzyme using the buffer conditions of Stack and Gray (19). Hydrolyses were initiated by the addition of SMEP1 to a solution of DNP-Pro-Leu-Gly-Leu-Trp-Ala-d-Arg-NH2 (100 'tM) in 100 ,L of assay buffer. The reaction was incubated at 370C and hydrolysis proceeded for 2.5 h. Aliquots were removed during the hydrolysis and the reaction quenched www.plantphysiol.org on April 3, 2019 -Published by Downloaded from

Construction and characterization of the soybean leaf metalloproteinase cDNA1

“…The suggestion that the 31 kDa protein represents the proenzyme is consistent with our previous finding [5] that the N-terminal amino acid of the mature SMEPl is Tyri34 and the observation that members of the matrix metalloproteinase family are synthesized in a prepro form with the proenzyme representing an inactive zymogen [2,15]. Examination of the amino acid sequence of the pro region reveals a stretch of 17 amino acids at positions 101-117 (Fig.…”

“…Our laboratory was the first to clearly demonstrate the presence of a metalloproteinase in higher plants [4,5]. A similar enzyme has been reported from the green alga, Chlamy-*Corresponding author.…”

“…Chemical sequencing of the entire mature form of the SMEP1 polypeptide revealed that the plant enzyme shared several biochemical features with the matrix metalloproteinase family of enzymes from vertebrate origin [5]. These features include a conserved zinc binding motif, an identical cleavage site within a synthetic collagenase-specific substrate, and similar inhibition constants when complexed with the tissue inhibitor of metalloproteinases [15].…”

“…We previously reported on the purification of the mature form of SMEPl [4] and the complete chemical se- quencing of the polypeptide [5]. This information was used to synthesize a set of degenerate oligonucleotide primers to amplify a portion of the SMEPl mRNA.…”

“…To generate a partial SMEP1 cDNA, two degenerate oligonucleotides derived from the primary amino acid sequence data of SMEP1 [5], were used in a polymerase chain reaction with ds cDNA synthesized with the Riboclone cDNA Synthesis System (Promega) to am-plify a 306 bp fragment encoding the first 102 amino acids of the mature polypeptide.…”