Characterization and purification of the discoidin domain-containing protein retinoschisin and its interaction with galactose.
RS1, also known as retinoschisin, is an extracellular discoidin domain-containing protein that has been implicated in maintaining the cellular organization and synaptic structure of the vertebrate retina. Mutations in the gene encoding RS1 are responsible for X-linked retinoschisis, a retinal degenerative disease characterized by the splitting of the retinal cell layers and visual impairment. To better understand the role of RS1 in retinal cell biology and X-linked retinoschisis, we have studied the interaction of wild-type and mutant RS1 with various carbohydrates coupled to agarose supports. RS1 bound efficiently to galactose-agarose and to a lesser extent lactose-agarose, but not agarose, N-acetylgalactosamine-agarose, N-acetylglucosamine-agarose, mannose-agarose, or heparin-agarose. RS1 cysteine mutants (C59S/C223S and C59S/C223S/C40S) which prevent disulfide-linked octamer formation exhibited little if any binding to galactose-agarose. The disease-causing R141H mutant bound galactose-agarose at levels similar to that of wild-type RS1, whereas the R141S mutant resulted in a marked reduction in the level of galactose-agarose binding. RS1 bound to galactose-agarose could be effectively displaced by incubation with isopropyl beta- d-1-thiogalactopyranoside (IPTG). This property was used as a basis to develop an efficient purification procedure. Anion exchange and galactose affinity chromatography was used to purify RS1 from the culture media of stably transformed Sf21 insect cells that express and secrete RS1. This cell expression and protein purification method should prove useful in the isolation of RS1 for detailed structure-function studies.
Dyka
Frank M
FM
Department of Biochemistry and Molecular Biology, Centre for Macular Research, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada.
Wu
Winco W H
WW
Pfeifer
Tom A
TA
Molday
Laurie L
LL
Grigliatti
Thomas A
TA
Molday
Robert S
RS
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D000070821:Discoidins; D005136:Eye Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism*; D006801:Humans; D037102:Lectins / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry*
D000070821:Discoidins; D005136:Eye Proteins; D037102:Lectins; D015800:Protozoan Proteins; C108726:RS1 protein, human; D005690:Galactose