Functional role of proteolytic processing of recombinant myocilin in self-aggregation.
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.
Aroca-Aguilar
José-Daniel
JD
Laboratorio de Genética Molecular Humana, Facultad de Medicina/Centro Regional de Investigaciones Biomédicas, Universidad de Castilla-La Mancha, Albacete, Spain.
Martínez-Redondo
Francisco
F
Sánchez-Sánchez
Francisco
F
Coca-Prados
Miguel
M
Escribano
Julio
J
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
D003598:Cytoskeletal Proteins; D004220:Disulfides; D005136:Eye Proteins; D006023:Glycoproteins; D011994:Recombinant Proteins; D000099179:Myocilin
Invest Ophthalmol Vis Sci. 2006 Jan;47(1):213-21
16384965
J Biol Chem. 2007 Sep 21;282(38):27810-24
17650508
Invest Ophthalmol Vis Sci. 2008 May;49(5):1932-9
18436825
J Immunol Methods. 1997 Jun 23;205(1):67-72
9236916
Ophthalmic Genet. 2000 Sep;21(3):155-69
11035548
Biochem Biophys Res Commun. 2006 Jan 20;339(3):797-804
16316624
Biochemistry. 1991 Sep 24;30(38):9143-53
1892825
FEBS Lett. 1997 Aug 18;413(2):349-53
9280311
Biochem J. 2005 Aug 1;389(Pt 3):675-84
15836428
Exp Eye Res. 2006 Dec;83(6):1386-95
16973161
Methods Mol Biol. 2004;261:167-74
15064457
Genomics. 1997 May 1;41(3):360-9
9169133
Invest Ophthalmol Vis Sci. 2000 Mar;41(3):729-40
10711688
J Biol Chem. 2007 Apr 6;282(14):10647-59
17293346
Anal Biochem. 1976 May 7;72:248-54
942051
Mol Vis. 2003 Nov 14;9:606-14
14627955
Mol Vis. 2004 Jun 27;10:417-25
15235575
Proc Natl Acad Sci U S A. 1993 May 15;90(10):4655-9
8506313
Prog Retin Eye Res. 2007 May;26(3):239-62
17321191
Mol Vis. 2007 Sep 13;13:1666-73
17893668
J Biochem. 1995 Nov;118(5):921-31
8749308
Mol Vis. 2002 Jul 22;8:271-9
12142865
Science. 1997 Jan 31;275(5300):668-70
9005853
Invest Ophthalmol Vis Sci. 2001 Apr;42(5):983-6
11274075
Prog Retin Eye Res. 1999 Jan;18(1):39-57
9920498
Dev Biol. 2001 Dec 15;240(2):340-60
11784068
Invest Ophthalmol Vis Sci. 2004 Oct;45(10):3560-7
15452063
Exp Eye Res. 2006 Jun;82(6):1017-29
16466712
Exp Eye Res. 2008 Oct;87(4):376-84
18674535
Invest Ophthalmol Vis Sci. 2002 Jan;43(1):151-61
11773026
Anal Biochem. 1979 Sep 15;98(1):231-7
94518
Biochem Biophys Res Commun. 2003 Mar 14;302(3):554-61
12615070
Invest Ophthalmol Vis Sci. 2001 Sep;42(10):2324-31
11527946
Hum Mol Genet. 1997 Nov;6(12):2091-7
9328473
Hum Mol Genet. 1999 May;8(5):899-905
10196380
J Biol Chem. 2005 Jun 3;280(22):21043-51
15795224