Characterization of Three Fragments That Constitute the Monomers of the Human Lysyl Hydroxylase Isoenzymes 1–3
2002; Elsevier BV; Volume: 277; Issue: 25 Linguagem: Inglês
10.1074/jbc.m112077200
ISSN1083-351X
AutoresKati Rautavuoma, Kati Takaluoma, Kaisa Passoja, Asta Pirskanen, Ari-Pekka Kvist, Kari I. Kivirikko, Johanna Myllyharju,
Tópico(s)Cancer-related gene regulation
ResumoLysyl hydroxylase (LH) catalyzes the formation of hydroxylysine in collagens; three human isoenzymes have been cloned so far. We report here on the purification of all three recombinant isoenzymes to homogeneity from the medium of cultured insect cells, and we demonstrate that they are all homodimers. Limited proteolysis experiments identified two main protease-sensitive regions in the monomers of about 80–85 kDa, corresponding to three fragments A–C (from the N to C terminus), with molecular masses of about 30, 37, and 16 kDa, respectively. Fragment A was found to play no role in LH activity as a recombinant B–C polypeptide constituted a fully active hydroxylase with K m values for cosubstrates and the peptide substrate that were identical to those of the full-length enzyme. LH3, but not LH1 and LH2, has also been reported recently (Heikkinen, J., Risteli, M., Wang, C., Latvala, J., Rossi, M., Valtavaara, M., and Myllylä, R. (2000) J. Biol. Chem. 275, 36158–36163) to possess collagen glucosyltransferase activity. We confirm this highly surprising finding here and extend it by demonstrating that LH3 may also possess trace amounts of collagen galactosyltransferase activity. All the glucosyltransferase and galactosyltransferase activity of LH3 was found to reside in fragment A, which played no role in the hydroxylase activity of the polypeptide. This fragment is about 55% identical and 80% similar to the corresponding fragments of LH1 and LH2. However, the levels of the glycosyltransferase activities are so low that they may be of little biological significance. It is thus evident that human tissues must have additional glycosyltransferases that are responsible for most of the collagen glycosylation in vivo. Lysyl hydroxylase (LH) catalyzes the formation of hydroxylysine in collagens; three human isoenzymes have been cloned so far. We report here on the purification of all three recombinant isoenzymes to homogeneity from the medium of cultured insect cells, and we demonstrate that they are all homodimers. Limited proteolysis experiments identified two main protease-sensitive regions in the monomers of about 80–85 kDa, corresponding to three fragments A–C (from the N to C terminus), with molecular masses of about 30, 37, and 16 kDa, respectively. Fragment A was found to play no role in LH activity as a recombinant B–C polypeptide constituted a fully active hydroxylase with K m values for cosubstrates and the peptide substrate that were identical to those of the full-length enzyme. LH3, but not LH1 and LH2, has also been reported recently (Heikkinen, J., Risteli, M., Wang, C., Latvala, J., Rossi, M., Valtavaara, M., and Myllylä, R. (2000) J. Biol. Chem. 275, 36158–36163) to possess collagen glucosyltransferase activity. We confirm this highly surprising finding here and extend it by demonstrating that LH3 may also possess trace amounts of collagen galactosyltransferase activity. All the glucosyltransferase and galactosyltransferase activity of LH3 was found to reside in fragment A, which played no role in the hydroxylase activity of the polypeptide. This fragment is about 55% identical and 80% similar to the corresponding fragments of LH1 and LH2. However, the levels of the glycosyltransferase activities are so low that they may be of little biological significance. It is thus evident that human tissues must have additional glycosyltransferases that are responsible for most of the collagen glycosylation in vivo. lysyl hydroxylase N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Lysyl hydroxylase (LH,1EC 1.14.11.4), a luminally oriented peripheral membrane protein within the endoplasmic reticulum, catalyzes the hydroxylation of lysine in collagens and more than 15 other proteins by the hydroxylation of -X-Lys-Gly- sequences, for which it requires Fe2+, 2-oxoglutarate, O2, and ascorbate (1Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Related Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar, 2Myllyharju J. Kivirikko K.I. Ann. Med. 2001; 33: 7-21Crossref PubMed Scopus (557) Google Scholar). The resulting hydroxylysine residues have two important functions. They are essential for the stability of the intermolecular collagen cross-links, and their hydroxy groups serve as attachment sites for carbohydrate units, either the monosaccharide galactose or the disaccharide glucosylgalactose (1Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Related Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar). The functions of the hydroxylysine-linked carbohydrate units are not fully understood, but in the case of fibril-forming collagens they influence the lateral packing of collagen molecules into fibrils and the fibril diameters (1Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Related Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar,3Notbohm H. Nokelainen M. Myllyharju J. Fietzek P.P. Müller P.K. Kivirikko K.I. J. Biol. Chem. 1999; 274: 8988-8992Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Hydroxylysine residues appear to play an especially critical role in the type IV collagens of basement membranes, because mutations in the gene for the only LH present in the nematode Caenorhabditis elegans, which is involved in the synthesis of basement membrane collagens, are embryonic lethal (4Norman K.R. Moerman D.L. Dev. Biol. 2000; 227: 690-705Crossref PubMed Scopus (48) Google Scholar). Three isoenzymes of human LH have been cloned so far (5Hautala T. Byers M.G. Eddy R.L. Shows T.B. Kivirikko K.I. Myllylä R. Genomics. 1992; 13: 62-69Crossref PubMed Scopus (115) Google Scholar, 6Yeowell H.N., Ha, V. Walker L.C. Murad S. Pinnell S.R. J. Invest. Dermatol. 1992; 99: 864-869Abstract Full Text PDF PubMed Google Scholar, 7Valtavaara M. Papponen H. Pirttilä A.-M. Hiltunen K. Helander H. Myllylä R. J. Biol. Chem. 1997; 272: 6831-6834Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 8Passoja K. Rautavuoma K. Ala-Kokko L. Kosonen T. Kivirikko K.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10482-10486Crossref PubMed Scopus (91) Google Scholar, 9Valtavaara M. Szpirer C. Szpirer J. Myllylä R. J. Biol. Chem. 1998; 273: 12881-12886Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 10Yeowell H. Walker L.C. Matrix Biol. 1999; 18: 179-187Crossref PubMed Scopus (75) Google Scholar); the sizes of the processed human LH1, LH2, and LH3 polypeptides are 709, 712, and 714 amino acid residues, respectively (5Hautala T. Byers M.G. Eddy R.L. Shows T.B. Kivirikko K.I. Myllylä R. Genomics. 1992; 13: 62-69Crossref PubMed Scopus (115) Google Scholar, 6Yeowell H.N., Ha, V. Walker L.C. Murad S. Pinnell S.R. J. Invest. Dermatol. 1992; 99: 864-869Abstract Full Text PDF PubMed Google Scholar, 7Valtavaara M. Papponen H. Pirttilä A.-M. Hiltunen K. Helander H. Myllylä R. J. Biol. Chem. 1997; 272: 6831-6834Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 8Passoja K. Rautavuoma K. Ala-Kokko L. Kosonen T. Kivirikko K.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10482-10486Crossref PubMed Scopus (91) Google Scholar, 9Valtavaara M. Szpirer C. Szpirer J. Myllylä R. J. Biol. Chem. 1998; 273: 12881-12886Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The highest degree of sequence identity was found within the catalytically important C-terminal regions, which contain the two histidines and one aspartate (11Pirskanen A. Kaimio A.-M. Myllylä R. Kivirikko K.I. J. Biol. Chem. 1996; 271: 9398-9402Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 12Myllyharju J. Kivirikko K.I. EMBO J. 1997; 16: 1173-1180Crossref PubMed Scopus (164) Google Scholar, 13Kivirikko K.I. Myllyharju J. Matrix Biol. 1998; 16: 357-368Crossref PubMed Scopus (235) Google Scholar) that provide the three ligands for the binding of Fe2+ to the catalytic site and the arginine that binds the C-5 carboxyl group of the 2-oxoglutarate (14Passoja K. Myllyharju J. Pirskanen A. Kivirikko K.I. FEBS Lett. 1998; 434: 145-148Crossref PubMed Scopus (32) Google Scholar). The catalytic subunit of the other main collagen hydroxylase, prolyl 4-hydroxylase, has a separate peptide substrate-binding domain that is distinct from the catalytic domain (15Myllyharju J. Kivirikko K.I. EMBO J. 1999; 18: 306-312Crossref PubMed Scopus (62) Google Scholar), but no data are available to indicate whether LH also has a separate peptide-binding domain. All three LH isoenzymes have been expressed as recombinant proteins in insect cells, and the recombinant enzymes present in crude insect cell extracts have been shown to hydroxylate lysine residues with similar kinetic properties (7Valtavaara M. Papponen H. Pirttilä A.-M. Hiltunen K. Helander H. Myllylä R. J. Biol. Chem. 1997; 272: 6831-6834Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 8Passoja K. Rautavuoma K. Ala-Kokko L. Kosonen T. Kivirikko K.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10482-10486Crossref PubMed Scopus (91) Google Scholar, 9Valtavaara M. Szpirer C. Szpirer J. Myllylä R. J. Biol. Chem. 1998; 273: 12881-12886Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 11Pirskanen A. Kaimio A.-M. Myllylä R. Kivirikko K.I. J. Biol. Chem. 1996; 271: 9398-9402Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Krol B.J. Murad S. Walker L.C. Marshall M.K. Clark W.L. Pinnell S.R. Yeowell H.N. J. Invest. Dermatol. 1996; 106: 11-16Abstract Full Text PDF PubMed Scopus (17) Google Scholar). Very surprisingly, however, recombinant LH3 was also found to possess collagen glucosyltransferase activity but not LH1 or LH2 (17Heikkinen J. Risteli M. Wang C. Latvala J. Rossi M. Valtavaara M. Myllylä R. J. Biol. Chem. 2000; 275: 36158-36163Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). No data are available on the level of this activity in the LH3 polypeptide nor on the possible biological significance of the finding (17Heikkinen J. Risteli M. Wang C. Latvala J. Rossi M. Valtavaara M. Myllylä R. J. Biol. Chem. 2000; 275: 36158-36163Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). It is also unknown whether this isoenzyme also possesses collagen galactosyltransferase activity, as the glucose residue is only transferred to a galactosylhydroxylysine residue (1Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Related Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar). The aim of the present work was to purify all three recombinant human LH isoenzymes to homogeneity and to study whether it is possible to identify protease-sensitive regions in the LH polypeptides that may correspond to domain boundaries and to express properly folded recombinant fragments for elucidating their properties. An additional important goal was to determine whether pure recombinant LH3 preparations have any collagen glucosyltransferase activity, what the level of any such activity might be, how this activity may be related to the domain structure of the enzyme, and whether the enzyme also has collagen galactosyltransferase activity. A full-length LH2 cDNA (7Valtavaara M. Papponen H. Pirttilä A.-M. Hiltunen K. Helander H. Myllylä R. J. Biol. Chem. 1997; 272: 6831-6834Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) was created here by amplifying two cDNA fragments by PCR, the first one corresponding to nucleotides 1–534 of the coding sequence, with aNotI site preceding the translation start codon, and the second one containing the coding sequence from nucleotide 512 to the translation stop codon followed by 210 bp of 3′-untranslated sequence and an XbaI site. These fragments were obtained from human placenta and pancreas Marathon-Ready cDNAs (CLONTECH), respectively. The LH2 cDNA fragments were digested with NotI-PstI andPstI-XbaI, respectively, and ligated toNotI-XbaI-digested pBluescript. ABamHI site and one cytosine and six histidine codons were inserted by PCR between the codons coding for the last amino acid of the signal peptide and the first amino acid of the processed LH polypeptide in the full-length cDNAs for LH1 (5Hautala T. Byers M.G. Eddy R.L. Shows T.B. Kivirikko K.I. Myllylä R. Genomics. 1992; 13: 62-69Crossref PubMed Scopus (115) Google Scholar), LH2, and LH3 (8Passoja K. Rautavuoma K. Ala-Kokko L. Kosonen T. Kivirikko K.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10482-10486Crossref PubMed Scopus (91) Google Scholar). The His6-LH1 cDNA was digested withBamHI-EcoRI; the His6-LH2 and His6-LH3 cDNAs were digested withBamHI-XbaI, and the inserts were ligated to pAcGP67A (BD PharMingen). The constructs thus contain a baculovirus GP67 signal sequence, a histidine tag, and cDNAs coding for the processed LH polypeptides. LH fragment constructs with an N-terminal histidine tag were generated by amplifying cDNA fragments corresponding to amino acids 1–262, 255–572, and 565–709 of LH1 and 1–266, 261–577, and 570–714 of LH3. Each fragment contained a BamHI site, one cytosine and six histidine codons in the 5′ end, and a translation stop codon and anEcoRI site in the 3′ end. Fragment pair constructs corresponding to amino acids 1–572 and 255–709 of LH1 and 1–577 and 261–714 of LH3 were generated in a similar way and ligated toBamHI-EcoRI-digested pAcGP67A (BD PharMingen). The sequences were verified by DNA sequencing on an automated device (ABI Prism 377, Applied Biosystems, Inc.) using BigDye or dRhodamine Terminator Cycle Sequencing Ready Reaction kits (PE Biosystems). The recombinant baculovirus vectors were cotransfected intoSpodoptera frugiperda Sf9 cells with modifiedAutographa californica nuclear polyhedrosis virus DNA (BD PharMingen) by calcium phosphate transfection, and the recombinant viruses were selected (18Crossen R. Gruenwald S. Baculovirus Expression Vector System, Instruction Manual. PharMingen, San Diego, CA1998Google Scholar). High Five insect cells (Invitrogen) were cultured as monolayers in TNM-FH medium (Sigma) supplemented with 10% fetal bovine serum (BioClear) or in suspension in Sf900IISFM serum-free medium (Invitrogen). The cells, seeded at a density of 5 × 106 cells/100-mm plate or 1 × 106/ml, were infected at a multiplicity of 5 with the viruses coding for the histidine-tagged full-length LH1, LH2, or LH3 or the LH1 and LH3 fragments and fragment pairs. The cells were harvested 48 h after infection, washed, and homogenized as described before (8Passoja K. Rautavuoma K. Ala-Kokko L. Kosonen T. Kivirikko K.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10482-10486Crossref PubMed Scopus (91) Google Scholar, 11Pirskanen A. Kaimio A.-M. Myllylä R. Kivirikko K.I. J. Biol. Chem. 1996; 271: 9398-9402Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Aliquots of the soluble fractions of the cell homogenates were analyzed by 8 or 12% SDS-PAGE under reducing conditions, and the cell pellets were further solubilized in 1% SDS and analyzed in the same manner. To purify the histidine-tagged full-length LH1, LH2, and LH3 and the LH1 and LH3 fragments and fragment pairs, the media from the suspension cultures were harvested by centrifugation at 2,500 rpm for 10 min, and samples were incubated with ProBond metal chelate affinity resin (Invitrogen) pre-equilibrated with 0.2 m NaCl, 0.1m glycine, 10 μm dithiothreitol, 1% glycerol, and 0.02 m Tris buffer, pH 7.5, at 4 °C overnight with gentle mixing. The amount of ProBond used was 2 ml per 100-ml medium sample. The ProBond resin was packed into a column and washed with 10 volumes of the above buffer, and the bound proteins were eluted with the same buffer containing 0.2 or 0.4 mimidazole. Fractions of 3 ml were collected and analyzed by 12% SDS-PAGE under reducing conditions. Fractions containing the full-length LH polypeptides or the fragments or fragment pairs were pooled, and the samples were passed through a PD-10 column (AmershamBiosciences) equilibrated with 0.2 m NaCl, 0.1m glycine, 10 μm dithiothreitol, 1% glycerol, and 0.02 m Tris buffer, pH 7.5, to remove the imidazole. The protein concentrations were measured with a Bio-Rad protein assay kit (Bio-Rad) according to the manufacturer's instructions. The molecular weights of the full-length LH isoenzymes were determined by gel filtration in a calibrated HiLoad 16/60 Superdex 200 column (Amersham Biosciences), equilibrated, and eluted with 0.2m NaCl, 0.1 m glycine, 1% glycerol, and 0.02m Tris buffer, pH 7.5. Purified full-length recombinant human LH1, LH2, or LH3, 10–100 μg, was digested with thermolysin, trypsin, or proteinase K at a protease:LH ratio of 1:100 to 1:10 at 37 °C for 30–90 min, and the digestion was stopped by the addition of Pefabloc SC (Roche Molecular Biochemicals) or EDTA (for thermolysin) to 3 mm. Samples were analyzed by 12% Tris-Tricine PAGE, transferred to a ProBlott™ membrane (Applied Biosystems, Inc.) by Western blotting, and stained with Coomassie Blue. The protease-resistant peptides were cut from the membrane and subjected to N-terminal sequencing in an Applied Biosystems, Inc., 477A pulse-liquid protein sequencer. LH activity was assayed by a method based on the hydroxylation-coupled decarboxylation of 2-oxo[1-14C]glutarate using a synthetic peptide (Ile-Lys-Gly)3 as the substrate (19Kivirikko K.I. Myllylä R. Methods Enzymol. 1982; 82: 245-304Crossref PubMed Scopus (325) Google Scholar). K m values were determined as described previously (20Puistola U. Turpeenniemi-Hujanen T.M. Myllylä R. Kivirikko K.I. Biochim. Biophys. Acta. 1980; 611: 40-50Crossref PubMed Scopus (60) Google Scholar). Collagen galactosyltransferase and glucosyltransferase activities were assayed by a method based on the transfer of [14C]galactose or [14C]glucose from radioactive UDP-galactose or UDP-glucose to hydroxylysine or galactosylhydroxylysine residues, respectively, in a denatured citrate-soluble rat skin collagen substrate, and the values were converted to mmol of the radioactive product synthesized per mol of enzyme in 1 min with a saturating concentration of the collagen substrate (19Kivirikko K.I. Myllylä R. Methods Enzymol. 1982; 82: 245-304Crossref PubMed Scopus (325) Google Scholar, 21Myllylä R. Risteli L. Kivirikko K.I. Eur. J. Biochem. 1975; 52: 401-410Crossref PubMed Scopus (89) Google Scholar). Purified LH polypeptides were analyzed by 8% non-denaturing PAGE. Transverse 0–8m urea gradient gel electrophoresis (22Wingfield P.T. Pain R.H. Coligan J.E. Dunn B.E. Ploegh H.L. Speicher D.W. Wingfield P.T. Current Protocols in Protein Science. John Wiley and Sons Inc., New York1998: 7.4.1-7.4.13Google Scholar) was carried out using 50 μg of purified LH polypeptides. CD spectrum measurements were carried out with a Jasco J500A spectropolarimeter in a 1-mm quartz cuvette. The use of a baculovirus signal peptide GP67 has been reported previously (16Krol B.J. Murad S. Walker L.C. Marshall M.K. Clark W.L. Pinnell S.R. Yeowell H.N. J. Invest. Dermatol. 1996; 106: 11-16Abstract Full Text PDF PubMed Scopus (17) Google Scholar) to lead to efficient secretion of a recombinant LH1 polypeptide expressed in insect cells. In order to develop a simple method for purifying LH polypeptides, cDNAs coding for the processed LH1, LH2, and LH3 polypeptides were cloned into a pAcGP67A expression vector, and a histidine tag was inserted between the GP67 signal sequence and the LH cDNA in each case. High Five insect cells cultured in suspension were infected with baculoviruses coding for the histidine-tagged LH1, LH2, or LH3 polypeptides. Medium samples were harvested 48 h after infection and incubated with a ProBond metal chelate affinity resin, and the unbound proteins were washed off with the column equilibration buffer, and the bound proteins were eluted with imidazole. The fractions were analyzed by SDS-PAGE and were found to contain polypeptides with sizes of 80–85 kDa, corresponding to full-length LH1, LH2, or LH3. The identities of the polypeptides were verified by N-terminal sequencing (data not shown). The fractions containing the LH1, LH2, or LH3 polypeptides were pooled; imidazole was removed in a PD-10 column, and the purified samples were analyzed by SDS-PAGE (Fig. 1 A) and nondenaturing PAGE (Fig. 1 B). The final preparations were found to be pure after one-step metal chelate affinity purification (Fig. 1) and to have LH activity (as shown for LH1 in Table I). Gel filtration experiments in a calibrated column indicated that all three LH isoenzymes had identical molecular weights, about 180,000 (details not shown).Table ILysyl hydroxylase activities of purified recombinant LH1 and its recombinant fragmentsPolypeptideLysyl hydroxylase activity dpm/nmolLH1 Expression set 1116,000 Expression set 294,000B–C Expression set 183,000 Expression set 2142,000A0A–B0The activity values obtained for various LH1 preparations varied considerably due to inactivation. Therefore, the values are shown for two separate sets of expression, purification, and assay (sets 1 and 2, respectively) of full-length LH1 and fragment pair B–C. All values are given as dpm of 14CO2 formed per nmol of polypeptide. Open table in a new tab The activity values obtained for various LH1 preparations varied considerably due to inactivation. Therefore, the values are shown for two separate sets of expression, purification, and assay (sets 1 and 2, respectively) of full-length LH1 and fragment pair B–C. All values are given as dpm of 14CO2 formed per nmol of polypeptide. In order to identify possible domain structures of the human LH1, LH2, and LH3 polypeptides, the purified recombinant enzymes were subjected to limited proteolysis with thermolysin, trypsin, or proteinase K for 30–90 min at 37 °C. The samples were analyzed by 12% Tris-Tricine PAGE and Coomassie staining. Five major protease-resistant peptides of about 68, 37, 33, 18, and 16 kDa were found after digestion of the LH polypeptides with thermolysin, trypsin, and proteinase K (as shown for a thermolysin-digested sample of LH1 in Fig.2), except that the 18-kDa fragment was not obtained for LH3 (data not shown). N-terminal sequencing of the major protease-resistant LH1 peptides showed that the N terminus of the 68- and 33-kDa peptides contained the histidine tag, whereas the N-terminal amino acid of the 37-kDa peptide was either Gly-261, Ile-262 (Fig.3 A), or Asp-253 (proteinase K, data not shown) depending on the protease used, and those of the 18- and 16-kDa peptides were either Ile-571 (Fig. 3 A) or Gly-573 (proteinase K). The N-terminal amino acids of the corresponding protease-resistant LH3 peptides were a histidine from the affinity tag, Leu-241 or Thr-264 and Leu-576 (Fig. 3 A) or Gly-578 (proteinase K, not shown). The data would be consistent with the presence of at least two main protease-sensitive regions in LH polypeptides and would suggest the existence of at least three structural domains, termed fragments A, B, and C, respectively (Fig.3 B). The calculated molecular masses with these cleavage sites are 29.6, 36.2, and 15.9 kDa for the nonglycosylated LH1 fragments and 30.4, 36.2, and 15.8 for the LH3 fragments. The 68-kDa polypeptide corresponds to fragment pair A-B, the 33-kDa polypeptide to fragment A with efficient utilization of its twoN-glycosylation sites, and the 37-kDa polypeptide represents fragment B, whereas the 18-kDa peptide obtained only from LH1 is probably due to utilization of the N-glycosylation site present at residue 668 in the 16-kDa fragment C of LH1 but not LH3. The N-terminal amino acids of the protease-resistant LH2 peptides were not determined, but in view of their similarity in size to the protease-resistant LH1 and LH3 peptides, these sites were assumed to be located in the corresponding regions. Protease cleavages at some additional sites were also occasionally observed in some of the purified LH samples, mainly when a 1:10 protease to LH ratio was used (Fig. 3 A). No such additional sites were identified with either thermolysin or trypsin within regions B and C (Fig. 3), whereas two additional protease-sensitive sites were found within region A in some experiments, but not all, and therefore this region may in fact consist of up to three domains (Fig. 3). However, as these minor sites were seen only in some experiments, they may also represent protease-sensitive sites within a folded domain. Limited proteolysis with proteinase K identified a few additional sites in some experiments that were not sensitive to thermolysin or trypsin and are therefore not likely to represent additional domain boundaries (details not shown). In order to study whether it is possible to express the recombinant LH1 and LH3 fragments A–C in folded forms, recombinant pAcGP67A vectors coding for the LH1 amino acids 1–262, 255–572, and 565–709 and the LH3 amino acids 1–266, 261–577, and 570–714 were generated. Each construct contained sequences coding for a histidine tag between those coding for the GP67 signal sequence and the LH fragments. Recombinant viruses coding for the LH fragments were used to infect High Five insect cells in suspension, and the cells and medium samples were harvested 48 h after infection. The cells were homogenized in a buffer containing 1% Nonidet P-40 and centrifuged, and the cell pellet was homogenized further in a buffer containing 50% glycerol (11Pirskanen A. Kaimio A.-M. Myllylä R. Kivirikko K.I. J. Biol. Chem. 1996; 271: 9398-9402Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), incubated on ice, and centrifuged. The remaining pellet was solubilized in 1% SDS, and the samples were analyzed by SDS-PAGE. All recombinant LH fragments were found to be partly soluble in the glycerol-containing buffer, but the highest amounts were found in the SDS-soluble fraction (details not shown). Similar data have been reported previously (8Passoja K. Rautavuoma K. Ala-Kokko L. Kosonen T. Kivirikko K.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10482-10486Crossref PubMed Scopus (91) Google Scholar, 11Pirskanen A. Kaimio A.-M. Myllylä R. Kivirikko K.I. J. Biol. Chem. 1996; 271: 9398-9402Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) for the full-length LH1 and LH3 polypeptides. Fragments A of LH1 and LH3 were secreted into the culture medium and could be purified to homogeneity from medium samples as described above for the full-length polypeptides (Fig.4 A, lanes 1 and 3). In contrast, the levels of fragments B and C in the culture medium were too low for attempts to purify them to homogeneity (details not shown). To study whether it is possible to express the LH1 and LH3 fragment pairs A-B and B-C in folded forms, recombinant pAcGP67A vectors coding for the LH1 amino acids 1–572 and 255–709 and the LH3 amino acids 1–577 and 261–714 were generated with N-terminal histidine tags, and the recombinant viruses were used to infect High Five insect cells in suspension. A significant portion of these fragment pairs was found to be secreted into the culture medium and could be purified to homogeneity as above (Fig. 4 A, lanes 2, 3, 5, and 6). To study whether the purified recombinant A fragments and the A-B and B-C pairs were folded, purified samples were analyzed by urea gradient gel electrophoresis and circular dichroism spectroscopy. Urea gradient gel analyses indicated that the recombinant LH1 and LH3 A fragments and fragment pairs were fully folded, as a sigmoidal transition in mobility resulting from unfolding along the urea gradient was observed in all cases, as shown in Fig. 4 B for LH1 fragment A. The far-UV CD spectra of the purified recombinant LH1 and LH3 A fragments and the A-B and B-C pairs were also typical of a folded protein (Fig.5). The purified recombinant full-length LH1 and LH3 isoenzymes and their recombinant fragments were analyzed for lysyl hydroxylase activity with an assay based on the hydroxylation-coupled decarboxylation of 2-oxo[1-14C]glutarate with the synthetic peptide (Ile-Lys-Gly)3 as a substrate (19Kivirikko K.I. Myllylä R. Methods Enzymol. 1982; 82: 245-304Crossref PubMed Scopus (325) Google Scholar). As reported previously (17Heikkinen J. Risteli M. Wang C. Latvala J. Rossi M. Valtavaara M. Myllylä R. J. Biol. Chem. 2000; 275: 36158-36163Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), the imidazole that was used to elute the nickel affinity column rapidly abolished the LH activity of the full-length enzymes and their fragment pairs, especially those of LH3. Consequently, activity values are given in Table I only for LH1. As expected, the recombinant LH1 and LH3 A fragments and A-B pairs that lacked the C-terminal catalytic region had no LH activity (as shown for LH1 in Table I). Surprisingly, the recombinant LH1 pair B-C, which contains all the critical residues required for cosubstrate binding and lacks the N-terminal 30-kDa A fragment, was a fully active lysyl hydroxylase (Table I). The LH3 pair B-C lost its activity particularly rapidly. The activity levels obtained with this fragment pair were nevertheless found in some purification experiments to be identical to those measured for the full-length LH3 purified at the same time (details not shown), indicating that the B-C pair of LH3 is probably also a fully active enzyme. The K m values of the LH1 B-C pair for the peptide substrate and the cosubstrates Fe2+, 2-oxoglutarate, and ascorbate were essentially identical to those of the full-length LH1 and LH3 enzymes (TableII).Table IIK m values of purified recombinant LH1 and LH3 and the recombinant LH1 fragment pair B–C for cosubstrates and peptide substrateSubstrate or cosubstrate K mLH1LH1:B–CLH3Fe2+5412-Oxoglutarate120140100Ascorbate350330350(IKG)3400380430K m values were determined as described previously (20Puistola U. Turpeenniemi-Hujanen T.M. Myllylä R. Kivirikko K.I. Biochim. Biophys. Acta. 1980; 611: 40-50Crossref PubMed Scopus (60) Google Scholar). Open table in a new tab K m values were determined as descr
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