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Role of Loop Structures of Neuropsin in the Activity of Serine Protease and Regulated Secretion

2002; Elsevier BV; Volume: 277; Issue: 17 Linguagem: Inglês

10.1074/jbc.m110725200

ISSN

1083-351X

Autores

Takuya Oka, Toshio Hakoshima, Makoto Itakura, Saori Yamamori, Masami Takahashi, Yasuhide Hashimoto, Sadao Shiosaka, Keiko Kato,

Tópico(s)

Axon Guidance and Neuronal Signaling

Resumo

Neuropsin involved in neural plasticity in adult mouse brain is a member of the S1 (clan SA) family of serine proteases and forms characteristic surface loops surrounding the substrate-binding site (Kishi, T., Kato, M., Shimizu, T., Kato, K., Matsumoto, K., Yoshida, S., Shiosaka, S., and Hakoshima, T. (1999)J. Biol. Chem. 274, 4220–4224). Little, however, is known about the roles of these loops. Thus, the present study investigated whether surface loop structures of neuropsin were essential for the generation of enzymatic activity and/or secretion of the enzyme via a regulated secretory pathway. The loops include those stabilized by six disulfide bonds or a loop C (Gly69–Glu80) and anN-glycosylated kallikrein loop (His91–Ile103) not containing a site linked by a disulfide bond. First, among the six disulfide bonds, only SS1 in loop E (Gly142–Leu155) and SS6 in loop G (Ser185–Gly197) were necessary for the catalytic efficiency of neuropsin. Second, disruptions of loop C and the N-linked oligosaccharide chain on the kallikrein loop affected the catalytic efficiency and P2 specificity, respectively. Alternatively, disruptions of loop C and the kallikrein loop enhanced the regulated secretion, whereas there was no one disruption that inhibited the secretion, indicating that there was no critical loop required for the regulated secretion among loops surrounding the substrate-binding site. Neuropsin involved in neural plasticity in adult mouse brain is a member of the S1 (clan SA) family of serine proteases and forms characteristic surface loops surrounding the substrate-binding site (Kishi, T., Kato, M., Shimizu, T., Kato, K., Matsumoto, K., Yoshida, S., Shiosaka, S., and Hakoshima, T. (1999)J. Biol. Chem. 274, 4220–4224). Little, however, is known about the roles of these loops. Thus, the present study investigated whether surface loop structures of neuropsin were essential for the generation of enzymatic activity and/or secretion of the enzyme via a regulated secretory pathway. The loops include those stabilized by six disulfide bonds or a loop C (Gly69–Glu80) and anN-glycosylated kallikrein loop (His91–Ile103) not containing a site linked by a disulfide bond. First, among the six disulfide bonds, only SS1 in loop E (Gly142–Leu155) and SS6 in loop G (Ser185–Gly197) were necessary for the catalytic efficiency of neuropsin. Second, disruptions of loop C and the N-linked oligosaccharide chain on the kallikrein loop affected the catalytic efficiency and P2 specificity, respectively. Alternatively, disruptions of loop C and the kallikrein loop enhanced the regulated secretion, whereas there was no one disruption that inhibited the secretion, indicating that there was no critical loop required for the regulated secretion among loops surrounding the substrate-binding site. Several serine proteases have been shown to play important roles in synaptic plasticity (1.Baranes D. Lederfein D. Huang Y.-Y. Chen M. Bailey C.H. Kandel E.R. Neuron. 1998; 21: 813-825Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 2.Komai S. Matsuyama T. Matsumoto K. Kato K. Kobayashi M. Imamura K. Yoshida S. Ugawa S. Shiosaka S. Eur. J. Neurosci. 2000; 12: 1479-1486Crossref PubMed Scopus (79) Google Scholar, 3.Davies B. Kearns I.R. Ure J. Davies C.H. Lathe R. J. Neurosci. 2001; 21: 6993-7000Crossref PubMed Google Scholar). These functions are suggested to be mediated by the activation of specific cell surface receptors and the degradation of extracellular matrix proteins and cell adhesion molecules (4.Nakagami Y. Abe K. Nishiyama N. Matsuki N. J. Neurosci. 2000; 20: 2003-2010Crossref PubMed Google Scholar, 5.Nicole O. Docagne F. Ali C. Margaill I. Carmeliet P. MacKenzie E.T. Vivien D. Buisson A. Nat. Med. 2001; 7: 59-64Crossref PubMed Scopus (621) Google Scholar). Neuropsin is a secretory serine protease expressed predominantly in pyramidal neurons in the hippocampal subfields CA1–3 (6.Chen Z.-L. Yoshida S. Kato K. Momota Y. Suzuki J. Tanaka T. Ito J. Nishino H. Aimoto S. Kiyama H. Shiosaka S. J. Neurosci. 1995; 15: 5088-5097Crossref PubMed Google Scholar) and is implicated in activity-dependent plasticity changes in neurons (2.Komai S. Matsuyama T. Matsumoto K. Kato K. Kobayashi M. Imamura K. Yoshida S. Ugawa S. Shiosaka S. Eur. J. Neurosci. 2000; 12: 1479-1486Crossref PubMed Scopus (79) Google Scholar, 3.Davies B. Kearns I.R. Ure J. Davies C.H. Lathe R. J. Neurosci. 2001; 21: 6993-7000Crossref PubMed Google Scholar, 6.Chen Z.-L. Yoshida S. Kato K. Momota Y. Suzuki J. Tanaka T. Ito J. Nishino H. Aimoto S. Kiyama H. Shiosaka S. J. Neurosci. 1995; 15: 5088-5097Crossref PubMed Google Scholar, 7.Momota Y. Yoshida S. Ito J. Shibata M. Kato K. Sakurai K. Matsumoto K. Shiosaka S. Eur. J. Neurosci. 1998; 10: 760-764Crossref PubMed Scopus (67) Google Scholar). The activity of neuropsin is regulated by a specific inhibitor, serine proteinase inhibitor-3, in adult mouse brain (8.Kato K. Kishi T. Kamachi T. Akisada M. Oka T. Midorikawa R. Takio K. Dohmae N. Bird P.I. Sun J. Scott F. Miyake Y. Yamamoto K. Machida A. Tanaka T. Matsumoto K. Shibata M. Shiosaka S. J. Biol. Chem. 2001; 276: 14562-14571Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The crystal structure of neuropsin has a serine protease fold that exhibits chimeric features of trypsin and nerve growth factor-γ, a member of the kallikrein family (Fig. 1 A) (9.Kishi T. Kato M. Shimizu T. Kato K. Matsumoto K. Yoshida S. Shiosaka S. Hakoshima T. J. Biol. Chem. 1999; 274: 4220-4224Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), both of which are a part of the S1 family (clan SA) of serine proteases (10.Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, London1998: 5-283Google Scholar). All S1 serine proteases possess two β-barrels with a catalytic His57 (chymotrypsin position number), Asp102, and Ser195 located at the interface of the two domains (10.Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, London1998: 5-283Google Scholar). These proteases, however, show diversity in the structures of surface loops surrounding the substrate-binding site, and it has been proposed that this diversity controls the specificity of enzymatic activity (9.Kishi T. Kato M. Shimizu T. Kato K. Matsumoto K. Yoshida S. Shiosaka S. Hakoshima T. J. Biol. Chem. 1999; 274: 4220-4224Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11.Hedstrom L. Szilagyi L. Rutter W.J. Science. 1992; 255: 1249-1253Crossref PubMed Scopus (454) Google Scholar, 12.Perona J.J. Craik C.S. Protein Sci. 1995; 4: 337-360Crossref PubMed Scopus (765) Google Scholar, 13.Perona J.J. Craik C.S. J. Biol. Chem. 1997; 272: 29987-29990Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 14.Huang C. Li L. Krilis S.A. Chanasyk K. Tang Y. Li Z. Hunt J.E. Stevens R.L. J. Biol. Chem. 1999; 274: 19670-19676Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Four to six disulfide bonds form, which probably provide a degree of structural rigidity to the loop structures (9.Kishi T. Kato M. Shimizu T. Kato K. Matsumoto K. Yoshida S. Shiosaka S. Hakoshima T. J. Biol. Chem. 1999; 274: 4220-4224Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 15.Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Crossref PubMed Scopus (648) Google Scholar,16.Renatus M. Engh R.A. Stubbs M.T. Huber R. Fischer S. Kohnert U. Bode W. EMBO J. 1997; 16: 4797-4805Crossref PubMed Scopus (94) Google Scholar). Neuropsin possesses six disulfide bonds, the same as trypsin. Furthermore, neuropsin forms a loop C (Gly69–Glu80) and an N-glycosylated loop D (17.Takahashi N. Tsukamoto Y. Shiosaka S. Kishi T. Hakoshima T. Arata Y. Yamaguchi Y. Kato K. Shimada I. Glycoconj. J. 1999; 16: 405-414Crossref PubMed Scopus (25) Google Scholar), the “kallikrein loop” (His91–Ile103), not containing a site linked by a disulfide bond. The loop C of neuropsin has been superimposed on that of trypsin and nerve growth factor-γ (9.Kishi T. Kato M. Shimizu T. Kato K. Matsumoto K. Yoshida S. Shiosaka S. Hakoshima T. J. Biol. Chem. 1999; 274: 4220-4224Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 18.Bartunik H.D. Summers L.J. Bartsch H.H. J. Mol. Biol. 1989; 210: 813-828Crossref PubMed Scopus (129) Google Scholar, 19.Bax B. Blundell T.L. Murray-Rust J. McDonald N.Q. Structure. 1997; 5: 1275-1285Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). On the other hand, the kallikrein loop is present in all members of the kallikrein family but not trypsin. However, the kallikrein loop of neuropsin differs radically from that of nerve growth factor-γ and of kallikrein (9.Kishi T. Kato M. Shimizu T. Kato K. Matsumoto K. Yoshida S. Shiosaka S. Hakoshima T. J. Biol. Chem. 1999; 274: 4220-4224Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 19.Bax B. Blundell T.L. Murray-Rust J. McDonald N.Q. Structure. 1997; 5: 1275-1285Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). While the loop of most members of the kallikrein family is cleaved into highly mobile nicked chains, that of neuropsin is packed without any nicked sites. This three-dimensional view of neuropsin provides the opportunity to examine the correlation between structure and substrate specificity (9.Kishi T. Kato M. Shimizu T. Kato K. Matsumoto K. Yoshida S. Shiosaka S. Hakoshima T. J. Biol. Chem. 1999; 274: 4220-4224Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), and it is necessary to evaluate the correlation experimentally. Most family S1 (clan SA) serine proteases are synthesized as precursors and then enter the secretory pathway (10.Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, London1998: 5-283Google Scholar). It has been indicated that some are sorted to a regulated secretory pathway (20.Burgess T.L. Craik C.S. Matsuuchi L. Kelly R.B. J. Cell Biol. 1987; 105: 659-668Crossref PubMed Scopus (44) Google Scholar, 21.Gullberg U. Lindmark A. Lindgren G. Persson A.-M. Nilsson E. Olsson I. J. Biol. Chem. 1995; 270: 12912-12918Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 22.Parmer R.J. Mahata M. Mahata S. Sebald M.T. O'Connor D.T. Miles L.A. J. Biol. Chem. 1997; 272: 1976-1982Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 23.Garwicz D. Lindmark A. Persson A.-M. Gullberg U. Blood. 1998; 92: 1415-1422Crossref PubMed Google Scholar). For example, the secretion of trypsinogen, elastase, and cathepsin G is clearly regulated (20.Burgess T.L. Craik C.S. Matsuuchi L. Kelly R.B. J. Cell Biol. 1987; 105: 659-668Crossref PubMed Scopus (44) Google Scholar, 21.Gullberg U. Lindmark A. Lindgren G. Persson A.-M. Nilsson E. Olsson I. J. Biol. Chem. 1995; 270: 12912-12918Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), whereas it is debatable whether kallikrein is secreted in a regulated manner (24.Sawyer N. Rondeau N. Chrétien M. Seidah N.G. DNA Cell Biol. 1991; 10: 259-269Crossref PubMed Scopus (6) Google Scholar, 25.Peters J. Takahashi S. Tada M. Miyake Y. J. Biochem. 1992; 111: 643-648Crossref PubMed Scopus (6) Google Scholar). However, it is not known which domain of these proteases is involved in regulated secretion. On the other hand, there is physiological evidence that neuropsin is involved in activity-dependent synaptic plasticity (2.Komai S. Matsuyama T. Matsumoto K. Kato K. Kobayashi M. Imamura K. Yoshida S. Ugawa S. Shiosaka S. Eur. J. Neurosci. 2000; 12: 1479-1486Crossref PubMed Scopus (79) Google Scholar, 3.Davies B. Kearns I.R. Ure J. Davies C.H. Lathe R. J. Neurosci. 2001; 21: 6993-7000Crossref PubMed Google Scholar, 6.Chen Z.-L. Yoshida S. Kato K. Momota Y. Suzuki J. Tanaka T. Ito J. Nishino H. Aimoto S. Kiyama H. Shiosaka S. J. Neurosci. 1995; 15: 5088-5097Crossref PubMed Google Scholar, 7.Momota Y. Yoshida S. Ito J. Shibata M. Kato K. Sakurai K. Matsumoto K. Shiosaka S. Eur. J. Neurosci. 1998; 10: 760-764Crossref PubMed Scopus (67) Google Scholar). However, it is still not clear whether neuropsin is secreted in response to stimuli. It is, thus, necessary to determine whether a secretagogue causes exocytotic release of neuropsin and, if so, which domain of neuropsin is required for regulated secretion. In the present study, site-directed removal involving six disulfide bonds, a loop C, and an N-glycosylated kallikrein loop of neuropsin was carried out, and the effects of these mutations on the enzymatic activity and the regulated secretion were investigated. pED1-NP was constructed as follows. A 789-bp NcoI-XhoI fragment of a full-length neuropsin cDNA was amplified based on NP1-pBluescript(II)KS+ (6.Chen Z.-L. Yoshida S. Kato K. Momota Y. Suzuki J. Tanaka T. Ito J. Nishino H. Aimoto S. Kiyama H. Shiosaka S. J. Neurosci. 1995; 15: 5088-5097Crossref PubMed Google Scholar) by PCR using the forward primer 5′-CGG GAT ATC ACT CAG CAT AAT G-3′ (T7 primer) and reverse primer 5′-GGA CTC GAG TCA GTC CCT GTT GTC CAT TGT CTT-3′ (primer-A, containing a stop codon and XhoI site) and introduced into the NcoI-XhoI site of pED1 vector (4896 bp) (a gift from Dr. Mahito Nakanishi, Gene Discovery Research Center, AIST, Ibaragi, Japan), which contains the cytomegalovirus enhancer, chicken β-actin promoter (26.Niwa H. Yamamura K. Miyazaki J. Gene (Amst.). 1991; 108: 193-200Crossref PubMed Scopus (4616) Google Scholar), and SV40 late poly(A) signal (27.Mizuguchi H. Nakagawa T. Nakanishi M. Imazu S. Nakagawa S. Mayumi T. Biochem. Biophys. Res. Commun. 1996; 218: 402-407Crossref PubMed Scopus (87) Google Scholar), to generate pED1-NP. Point mutations were introduced into a full-size neuropsin cDNA of pED1-NP by oligonucleotide-directed mutagenesis using Mutan-Super Express K m according to the manufacturer's protocol (TaKaRa, Siga, Japan). The numerals in the clone names indicate the amino acid number counted from the start codon, Met. The following primers were used, and the nucleotides changed relative to the neuropsin cDNA sequence are underlined: C7S, 5′-CCC CCA CCC TCT GCA ATC CA-3′; C39S, 5′-AGG TCG AGA GTC TAT ACC CCA C-3′; C74S, 5′-AGC CCA CTC CAA AAA ACA G-3′; C108S, 5′-GCA TCC TTC CTA CAA CAA C-3′; C145S, 5′-CCA ATC TGTCTC CCA AAG TTG GCC AGA AG-3′; C152S, 5′-TTG GCC AGA AGTCCA TCA TAT CAG G-3′; C198S, 5′-AGG GCA TGG TCTCTG CTG GCA GCA G-3′; C208S, 5′-TGA CAC GTC CCA GGG TG-3′; C233S, 5′-TCA GAC CCC TCT GGG AAA CCC G-3′; C246S, 5′-ACA CCA AAA TCT CCC GCT ACA CTA CC-3′; N110A, 5′-TCC TTG CTA CGC CAA CAG CAA CCC-3′; D206V, 5′-TGG AGC TGT CAC GTG CC-3′; DS211VA, 5′-TGG AGC TGA CAC GTG CCA GGG TGT CGC AGG AGG CCC-3′. Deletion mutants of Δ(S87-P94) and N110S·Δ(N113-E115) were created by PCR with NP1-pBluescript(II)KS+ (6.Chen Z.-L. Yoshida S. Kato K. Momota Y. Suzuki J. Tanaka T. Ito J. Nishino H. Aimoto S. Kiyama H. Shiosaka S. J. Neurosci. 1995; 15: 5088-5097Crossref PubMed Google Scholar). PCR fragments of 250 bp ofNcoI-EcoO65I (forward, T7 primer; reverse, 5′-ATGGTC ACC CAG ACG CAC G-3′) and 509 bp ofEcoO65I-XhoI (forward, 5′-TAC TCC GTG CGT CTG GGT GAC CAT GAG CAG GAG ATC CAG GTG GC-3′; reverse, primer-A) were inserted into the NcoI-XhoI site of pED1 to generate Δ(S87-P94). PCR fragments of 328 bp ofNcoI-NspV (forward, T7 primer; reverse, 5′-GTTCGAATA GCA AGG ATG CTG GAT AGA-3′) and 446 bp of NspV-XhoI (forward, 5′-TCT ATC CAG CAT CCT TGC TATTCG AAC AGC GAT CAC AGT CAC GAT ATA ATG-3′; reverse, primer-A) were inserted into theNcoI-XhoI site of pED1 to generate N110S·Δ(N113-E115). pSlhGH vector encoding human growth hormone was described previously (28.Itakura M. Misawa H. Sekiguchi M. Takahashi S. Takahashi M. Biochem. Biophys. Res. Commun. 1999; 265: 691-696Crossref PubMed Scopus (64) Google Scholar). The Neuro2a cell line (mouse neuroblastoma; Institute for Fermentation, Osaka, Japan) was grown in Eagle's medium (Nissui, Tokyo, Japan) supplemented with 1% nonessential amino acids (Invitrogen) and 10% fetal bovine serum at 37 °C in a 5% CO2 incubator. Neuro2a cells were plated at a density of 1.0 × 104/cm2(1.0 × 105 cells/35-mm dish) in Eagle's medium, 10% FCS, and 1% nonessential amino acids on a coverslip (Matsunami, Osaka, Japan) coated with poly-l-lysine (4 μg/cm2; Sigma). After 18 h, cells were transfected with 1 μg of pED1-NP and the mutants in OPTI-MEM plus 1% nonessential amino acids by LipofectAMINE PLUS (Invitrogen). After 3 h, the medium was replaced with fresh OPTI-MEM plus 1% nonessential amino acids containing N-2 supplement (differentiation) (Invitrogen). Conditioned medium (2 ml/35-mm dish) was recovered after 36 h of culture for the enzyme assay. The PC12 cell line (a gift from Dr. Yasuhisa Hukui, University of Tokyo, Japan) was grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 5% fetal bovine serum and 5% heat-inactivated horse serum at 37 °C in a 10% CO2incubator. PC12 cells were plated at a density of 1.2 × 105 cells/cm2 (1.2 × 106cells/35-mm dish) in Dulbecco's modified Eagle's medium, 5% fetal bovine serum, and 5% horse serum on a dish coated with polyethyleneimine (Sigma). After 18 h, cells were co-transfected with 1.5 μg of pSIhGH and 1.5 μg of pED1-neuropsin by LipofectAMINE 2000 (Invitrogen). After 48 h, assays of hGH and neuropsin release were performed. Media and cell lysates were subjected to SDS-PAGE using 10% acrylamide gel. The proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membrane was reacted with rabbit anti-neuropsin polyclonal antibody (11pAb) (8.Kato K. Kishi T. Kamachi T. Akisada M. Oka T. Midorikawa R. Takio K. Dohmae N. Bird P.I. Sun J. Scott F. Miyake Y. Yamamoto K. Machida A. Tanaka T. Matsumoto K. Shibata M. Shiosaka S. J. Biol. Chem. 2001; 276: 14562-14571Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) and then goat anti-rabbit IgG conjugated with alkaline phosphatase (Bio-Rad) in 5% skim milk in 0.1 m Tris-HCl, pH 7.5, 0.15 mNaCl, and 0.1% Tween-20. The secondary antibody was detected by enhanced chemiluminescence (Immun-Star Substrate, Bio-Rad), followed by exposure to x-ray film. The amount of mutant and wild-type protein was determined based on the band densities using one-dimensional gel image analysis software (Quantity One software, PDI, Toyobo Co., Ltd., Osaka, Japan) (8.Kato K. Kishi T. Kamachi T. Akisada M. Oka T. Midorikawa R. Takio K. Dohmae N. Bird P.I. Sun J. Scott F. Miyake Y. Yamamoto K. Machida A. Tanaka T. Matsumoto K. Shibata M. Shiosaka S. J. Biol. Chem. 2001; 276: 14562-14571Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Recombinant neuropsin (Baculo), purified from a baculovirus expression system (29.Shimizu C. Yoshida S. Shibata M. Kato K. Momota Y. Matsumoto K. Shiosaka T. Midorikawa R. Kamachi T. Kawabe A. Shiosaka S. J. Biol. Chem. 1998; 273: 11189-11196Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), was used as a control of the amount. For comparison of the activity of 11pAb to bind mutants and wild-type neuropsin, conditioned medium derived from each transfectant was immunoprecipitated with Affi-Gel Hz beads (Bio-Rad) conjugated with F12mAb (7.Momota Y. Yoshida S. Ito J. Shibata M. Kato K. Sakurai K. Matsumoto K. Shiosaka S. Eur. J. Neurosci. 1998; 10: 760-764Crossref PubMed Scopus (67) Google Scholar), the beads were subjected to reducing SDS-PAGE, and the band density stained using colloidal properties of Coomassie G-250 (Gelcode blue; Pierce) was compared with the band density obtained by Western blotting with 11pAb (8.Kato K. Kishi T. Kamachi T. Akisada M. Oka T. Midorikawa R. Takio K. Dohmae N. Bird P.I. Sun J. Scott F. Miyake Y. Yamamoto K. Machida A. Tanaka T. Matsumoto K. Shibata M. Shiosaka S. J. Biol. Chem. 2001; 276: 14562-14571Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). There was no difference in the band density of mutants and wild-type neuropsin by Western blotting with 11pAb per μg of protein contents (data not shown). The amidolytic activity of neuropsin was determined basically as described previously (29.Shimizu C. Yoshida S. Shibata M. Kato K. Momota Y. Matsumoto K. Shiosaka T. Midorikawa R. Kamachi T. Kawabe A. Shiosaka S. J. Biol. Chem. 1998; 273: 11189-11196Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Briefly, conditioned media of Neuro2a cells transfected with mutants and wild-type neuropsin were treated with lysyl endopeptidase (EC 3.4.21.50) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) conjugated with Sepharose 4B (Amersham Biosciences) at 37 °C for 15 min, and then the amount of mutant and wild-type neuropsin in the medium was determined based on the band density, following reducing SDS-PAGE and immunoblotting. To determine the amidolytic activity, 12.5–200 mmBoc-Val-Pro-Arg-4-methylcoumaryl-7-amide (MCA), Pro-Phe-Arg-MCA, Boc-Phe-Ser-Arg-MCA, and Boc-Asp (benzyloxy)-Pro-Arg-MCA (Peptide Institute, Inc., Osaka, Japan) were mixed with 50 nm mutant and wild-type neuropsin in 96-well plates (Corning Costar, Tokyo, Japan). The reaction proceeded at 25 °C for 0–60 min at 3-min intervals in 50 mm Tris-HCl, pH 8.0, 0.1 mg/ml bovine serum albumin, and 0.02% NaN3 and was monitored with a multiwell plate reader (Cytofluor II, PerSeptive Biosystems, Tokyo, Japan). The kinetic parameters K mand k cat were obtained by linear regression analysis of the Lineweaver-Burk plot. After 48 h of gene transfection, PC12 cells were washed four times with a low K+ solution (140 mm NaCl, 4.7 mmKCl, 1.2 mm KH2PO4, 2.5 mm CaCl2, 1.2 mm MgSO4, 11 mm glucose, and 15 mm HEPES-NaOH, pH 7.4). They were incubated for 2 min with the low K+ solution and then for 2 min with a high K+ solution (140 mmNaCl, 59.7 mm KCl, 1.2 mmKH2PO4, 2.5 mm CaCl2, 1.2 mm MgSO4, 11 mm glucose, and 15 mm HEPES-NaOH, pH 7.4), and each medium was recovered. Cell lysates were homogenized in 10 mm HEPES-NaOH, pH 7.4, and 0.2 mm EDTA using a sonicator (Ultrasonic Homogenizer, VP-5S, TAITEC, Co., Ltd., Saitama, Japan) and centrifuged at 17,500 × g for 5 min, and then the supernatant was collected. Contents of human growth hormone (hGH) were determined using an hGH enzyme-linked immunosorbent assay kit (Roche Molecular Biochemicals, Mannheim, Germany) (28.Itakura M. Misawa H. Sekiguchi M. Takahashi S. Takahashi M. Biochem. Biophys. Res. Commun. 1999; 265: 691-696Crossref PubMed Scopus (64) Google Scholar). Mutants and wild-type neuropsin were concentrated by acid precipitation with incubation in 6% trichloroacetic acid, 0.0125% deoxycholic acid, and 100 μg/ml gelatin on ice for 1.5 h and centrifugation at 17,500 ×g for 45 min, and then their content was determined by SDS-PAGE followed by Western blotting with 11pAb. Secretion was expressed as a percentage of total cellular hGH, wild-type neuropsin, and the mutants. The following antibodies were used in immunofluorescence: rabbit anti-neuropsin polyclonal antibody (11pAb, 20 μg/ml), rat anti-neuropsin monoclonal antibody (B5mAb, 20 μg/ml, Medial and Biological Laboratories Co., Ltd., Nagoya, Japan) (7.Momota Y. Yoshida S. Ito J. Shibata M. Kato K. Sakurai K. Matsumoto K. Shiosaka S. Eur. J. Neurosci. 1998; 10: 760-764Crossref PubMed Scopus (67) Google Scholar), mouse anti-Grp78 monoclonal antibody (diluted 1:200; StressGen Biotechnologies Corp., Victoria, BC, Canada) (30.Huovila A.-P.J. Eder A.M. Fuller S.D. J. Cell Biol. 1992; 118: 1305-1320Crossref PubMed Scopus (230) Google Scholar), rabbit antiserum against α-mannosidase II, a Golgi enzyme, (1:1,000, a gift from Dr. Kelly Moremen, University of Georgia, Athens, GA) (31.Moremen K.W. Robbins P.W. J. Cell Biol. 1991; 115: 1521-1534Crossref PubMed Scopus (118) Google Scholar), and rabbit anti-chromogranin A polyclonal antibody (1:500, a gift from Dr. Seung Hyun Yoo, KAIST, Korea) (32.Yoo S.H. Lim D.J. FEBS Lett. 1993; 317: 113-117Crossref PubMed Scopus (3) Google Scholar) as primary antibodies and goat anti-rabbit IgG conjugated with fluorescein isothiocyanate (1:600, BIOSOURCE International, Camarillo, CA), goat anti-rat IgG conjugated with rhodamine (1:100; BIOSOURCE International), and goat anti-mouse IgG conjugated with fluorescein isothiocyanate (1:600; BIOSOURCE International) as secondary antibodies. Cells were fixed with 4% paraformaldehyde in Dulbecco's phosphate-buffered saline containing 0.7 mmCaCl2 and 0.5 mm MgCl2, at 4 °C for 1 h and permeabilized with 0.2% Triton X-100, 20 mm glycine, and phosphate-buffered saline at 37 °C for 10 min. After a wash with 0.15 m NaCl, 20 mmboric acid, and 5 mm sodium tetraborate decahydrate, pH 8.0 (BBS), cells were incubated with BBS containing 3% bovine serum albumin at room temperature for 30 min and then with primary antibodies in BBS containing 3% bovine serum albumin at 4 °C overnight. After a wash with BBS, secondary antibodies in BBS containing 3% bovine serum albumin were applied at 4 °C overnight. After a wash with BBS, coverslips were mounted in glycerol-containing Mowiol 4–88 (Calbiochem-Novabiochem) and 1,4-diazobicyclo-(2,2,2)-octane (Sigma) (33.Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 418Google Scholar) and were observed with a Laser scan microscope LSM510 invert (Carl Zeiss, Tokyo, Japan). Double-labeled immunofluorescence cytochemistry with anti-Grp78, anti-α-mannosidase II, and anti-chromogranin A antibodies was used to observe the localization of mutant and wild-type neuropsin on the endoplasmic reticulum (30.Huovila A.-P.J. Eder A.M. Fuller S.D. J. Cell Biol. 1992; 118: 1305-1320Crossref PubMed Scopus (230) Google Scholar), Golgi complex (31.Moremen K.W. Robbins P.W. J. Cell Biol. 1991; 115: 1521-1534Crossref PubMed Scopus (118) Google Scholar), and large dense core vesicles (34.Kim T. Tao-Cheng J.-H. Eiden L.E. Loh Y.P. Cell. 2001; 106: 499-509Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar), respectively. To investigate the role of the surface loop structure of neuropsin in enzymatic activity and secretion, site-directed mutagenesis was employed in loop C (Gly69–Glu80), theN-glycosylated kallikrein loop (His91–Ile103), and six disulfide bonds (Fig. 1 B). Neuro2a cells transiently transfected with mutant and wild-type neuropsin cDNA were cultured for 36 h. Disruption of the disulfide bonds SS2, SS4, and SS5 interrupted the secretion and caused the enzymes to distribute in the endoplasmic reticulum but not the Golgi complex (data not shown). Since the results show quality control of the enzymes, they were uninformative with regard to the enzymatic activity and secretion. Twelve mutants and the wild-type neuropsin had little amidolytic activity without treatment by lysyl endopeptidase (data not shown) (29.Shimizu C. Yoshida S. Shibata M. Kato K. Momota Y. Matsumoto K. Shiosaka T. Midorikawa R. Kamachi T. Kawabe A. Shiosaka S. J. Biol. Chem. 1998; 273: 11189-11196Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Table I shows the enzymatic activities of the mutants and wild type detected after treatment with lysyl endopeptidase.Table IEnzymatic activity of mutants and wild type of neuropsin in media of transfected neuro2a cellsk catK mk cat/K ms−1μmm−1s−1Boc-Val-Pro-Arg-MCA Neuropsin (Baculo)1-aNeuropsin prepared from baculovirus expression system (28).68.82452.81 × 105 Wild type1002823.55 × 105 D206V0.14181.91.72 × 103 DS211VA0.038786.34.48 × 102 Δ(S87-P94)15.89661.64 × 104 N110S · Δ(N113-E115)51.697.15.32 × 105 N110A45.01522.96 × 105 C7S47.01902.48 × 105 C108S78.31584.95 × 105 C39S (SS1)11.67031.65 × 104 C145S (SS3)50.81104.60 × 105 C208S (SS6)0.5131025.03 × 103 C233S (SS6)0.4242122.00 × 103 C246S (SS3)28.81192.43 × 105Pro-Phe-Arg-MCA Wild type19383602.31 × 104 N110S · Δ(N113-E115)49.912703.92 × 104 N110A18.34104.47 × 104Boc-Phe-Ser-Arg-MCA Wild type34.82161.61 × 105 C145S (SS3)38.12091.82 × 105 C246S (SS3)31.42381.32 × 105 N110S · Δ(N113-E115)35.72151.66 × 105 N110A24.51801.36 × 105Boc-Asp(benzyloxy)-Pro-Arg-MCA Wild type31.33169.92 × 104 C145S (SS3)33.13091.07 × 105 C246S (SS3)22.72877.90 × 1041-a Neuropsin prepared from baculovirus expression system (28.Itakura M. Misawa H. Sekiguchi M. Takahashi S. Takahashi M. Biochem. Biophys. Res. Commun. 1999; 265: 691-696Crossref PubMed Scopus (64) Google Scholar). Open table in a new tab First, the enzymatic activity was examined with Boc-Val-Pro-Arg-MCA. Mutations in Asp189 (D206V; S1-specific pocket) and Ser195 (DS211VA; catalytic triad) lacking a protease active pocket resulted in levels of activity ∼200- and ∼800-fold less than the wild type as measured byk cat/K m, respectively (Table I, lines 3 and 4). Alternatively, C208S (SS6) and C233S (SS6), had 70–200-fold less activity than the wild type (Table I, lines 12 and 13), the k cat values being ∼200-fold less than and the K m values almost the same as the wild-type values. Disruptions of loop C (Δ(S87-P94)) and of a disulfide bond SS1 (C39S) caused hydration of the substrate to occur 22 times more slowly than for the wild-type (Table I, lines 5 and 10). Both loop C (Δ(S87-P94)) and C39S (SS1) showed a decrease ink cat values and increase inK m values, indicating that loop C (Gly69–Glu80) and SS1 were necessary for catalytic efficiency. The remaining mutants showed little difference in activity on Boc-Val-Pro-Arg-MCA, relative to the wild-type. The three-dimensional view has revealed that the kallikrein loop of neuropsin forms a narrow P2 pocket (9.Kishi T. Kato M. Shimizu T. Kato K. Matsumoto K. Yoshida S. Shiosaka S. Hakoshima T. J. Biol. Chem. 1999; 274: 4220-4224Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). To determine the effect ofN-glycosylation of the kallikrein loop on the P2 specificity of neuropsin experimentally, the activities of N110A and N110S·Δ(N113-E115) were examined with Pro-P

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