title: Functional role of proteolytic processing of recombinant myocilin in self-aggregation.
abstract: OBJECTIVE
  Recombinant myocilin expressed in cells in culture is endoproteolytically cleaved in the endoplasmic reticulum by calpain II, releasing an N-terminal and a C-terminal fragment. This proteolytic processing has been speculated to regulate the molecular interactions of myocilin. The main purpose of this study was to analyze the effect of the proteolytic cleavage on myocilin aggregation.
  
  
  METHODS
  cDNAs encoding human myocilin and the N- and C-terminal fragments were transiently expressed in HEK-293T cells. Covalent interactions of recombinant myocilin were analyzed by SDS-PAGE and Western immunoblot analysis in different dissociating conditions. Noncovalent interactions were studied by solid-phase binding assays, performed with Ni-chelating HPLC-purified recombinant proteins, and by Far-Western blot analysis.
  
  
  RESULTS
  Western blot analysis of recombinant myocilin aggregates under either increasing ionic strength or increasing concentration of reducing agent indicated that ionic interactions do not contribute to the stability of the molecular complexes linked by disulfide bridges. Disulfide myocilin homoaggregates decreased as the proteolytic processing increased. Solid-phase binding assays showed the existence of high-affinity (K(d) = 0.068 microM) noncovalent myocilin-myocilin interactions and that processed fragments bound to the full-length protein with significantly reduced affinity. Far-Western blot analysis confirmed noncovalent interactions between recombinant myocilin disulfide aggregates.
  
  
  CONCLUSIONS
  The proteolytic processing of recombinant myocilin decreases myocilin homoaggregates. These data provide the first evidence of a functional role for this processing in myocilin aggregation and suggest that disulfide complexes of myocilin could organize into a dynamic extracellular network sustained by noncovalent N-terminal interactions.
authors.affiliation: Laboratorio de Genética Molecular Humana, Facultad de Medicina/Centro Regional de Investigaciones Biomédicas, Universidad de Castilla-La Mancha, Albacete, Spain.
authors.forename: José-Daniel
authors.initials: JD
authors.lastname: Aroca-Aguilar
authors.forename: Francisco
authors.initials: F
authors.lastname: Martínez-Redondo
authors.forename: Francisco
authors.initials: F
authors.lastname: Sánchez-Sánchez
authors.forename: Miguel
authors.initials: M
authors.lastname: Coca-Prados
authors.forename: Julio
authors.initials: J
authors.lastname: Escribano
mesh_terms: D000818:Animals; D000869:Anterior Eye Segment / Q000378:metabolism; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000196:embryology; D009994:Osmolar Concentration; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry*; D012590:Sclera / Q000378:metabolism; D014162:Transfection; D000099179:Myocilin
chemical_list: D003598:Cytoskeletal Proteins; D004220:Disulfides; D005136:Eye Proteins; D006023:Glycoproteins; D011994:Recombinant Proteins; D000099179:Myocilin
references.citation: Invest Ophthalmol Vis Sci. 2006 Jan;47(1):213-21
references.pmid: 16384965
references.citation: J Biol Chem. 2007 Sep 21;282(38):27810-24
references.pmid: 17650508
references.citation: Invest Ophthalmol Vis Sci. 2008 May;49(5):1932-9
references.pmid: 18436825
references.citation: J Immunol Methods. 1997 Jun 23;205(1):67-72
references.pmid: 9236916
references.citation: Ophthalmic Genet. 2000 Sep;21(3):155-69
references.pmid: 11035548
references.citation: Biochem Biophys Res Commun. 2006 Jan 20;339(3):797-804
references.pmid: 16316624
references.citation: Biochemistry. 1991 Sep 24;30(38):9143-53
references.pmid: 1892825
references.citation: FEBS Lett. 1997 Aug 18;413(2):349-53
references.pmid: 9280311
references.citation: Biochem J. 2005 Aug 1;389(Pt 3):675-84
references.pmid: 15836428
references.citation: Exp Eye Res. 2006 Dec;83(6):1386-95
references.pmid: 16973161
references.citation: Methods Mol Biol. 2004;261:167-74
references.pmid: 15064457
references.citation: Genomics. 1997 May 1;41(3):360-9
references.pmid: 9169133
references.citation: Invest Ophthalmol Vis Sci. 2000 Mar;41(3):729-40
references.pmid: 10711688
references.citation: J Biol Chem. 2007 Apr 6;282(14):10647-59
references.pmid: 17293346
references.citation: Anal Biochem. 1976 May 7;72:248-54
references.pmid: 942051
references.citation: Mol Vis. 2003 Nov 14;9:606-14
references.pmid: 14627955
references.citation: Mol Vis. 2004 Jun 27;10:417-25
references.pmid: 15235575
references.citation: Proc Natl Acad Sci U S A. 1993 May 15;90(10):4655-9
references.pmid: 8506313
references.citation: Prog Retin Eye Res. 2007 May;26(3):239-62
references.pmid: 17321191
references.citation: Mol Vis. 2007 Sep 13;13:1666-73
references.pmid: 17893668
references.citation: J Biochem. 1995 Nov;118(5):921-31
references.pmid: 8749308
references.citation: Mol Vis. 2002 Jul 22;8:271-9
references.pmid: 12142865
references.citation: Science. 1997 Jan 31;275(5300):668-70
references.pmid: 9005853
references.citation: Invest Ophthalmol Vis Sci. 2001 Apr;42(5):983-6
references.pmid: 11274075
references.citation: Prog Retin Eye Res. 1999 Jan;18(1):39-57
references.pmid: 9920498
references.citation: Dev Biol. 2001 Dec 15;240(2):340-60
references.pmid: 11784068
references.citation: Invest Ophthalmol Vis Sci. 2004 Oct;45(10):3560-7
references.pmid: 15452063
references.citation: Exp Eye Res. 2006 Jun;82(6):1017-29
references.pmid: 16466712
references.citation: Exp Eye Res. 2008 Oct;87(4):376-84
references.pmid: 18674535
references.citation: Invest Ophthalmol Vis Sci. 2002 Jan;43(1):151-61
references.pmid: 11773026
references.citation: Anal Biochem. 1979 Sep 15;98(1):231-7
references.pmid: 94518
references.citation: Biochem Biophys Res Commun. 2003 Mar 14;302(3):554-61
references.pmid: 12615070
references.citation: Invest Ophthalmol Vis Sci. 2001 Sep;42(10):2324-31
references.pmid: 11527946
references.citation: Hum Mol Genet. 1997 Nov;6(12):2091-7
references.pmid: 9328473
references.citation: Hum Mol Genet. 1999 May;8(5):899-905
references.pmid: 10196380
references.citation: J Biol Chem. 2005 Jun 3;280(22):21043-51
references.pmid: 15795224