Proteomic analysis of potential keratan sulfate, chondroitin sulfate A, and hyaluronic acid molecular interactions.
OBJECTIVE
Corneal stroma extracellular matrix (ECM) glycosaminoglycans (GAGs) include keratan sulfate (KS), chondroitin sulfate A (CSA), and hyaluronic acid (HA). Embryonic corneal keratocytes and sensory nerve fibers grow and differentiate according to chemical cues they receive from the ECM. This study asked which of the proteins that may regulate keratocytes or corneal nerve growth cone immigration interact with corneal GAGs.


METHODS
Biotinylated KS (bKS), CSA (bCSA), and HA (bHA) were prepared and used in microarray protocols to assess their interactions with 8268 proteins and a custom microarray of 85 extracellular epitopes of nerve growth-related proteins. Surface plasmon resonance (SPR) was performed with bKS and SLIT2, and their ka, kd, and KD were determined.


RESULTS
Highly sulfated KS interacted with 217 microarray proteins, including 75 kinases, several membrane or secreted proteins, many cytoskeletal proteins, and many nerve function proteins. CSA interacted with 24 proteins, including 10 kinases and 2 cell surface proteins. HA interacted with 6 proteins, including several ECM-related structural proteins. Of 85 ECM nerve-related epitopes, KS bound 40 proteins, including SLIT, 2 ROBOs, 9 EPHs, 8 Ephrins (EFNs), 8 semaphorins (SEMAs), and 2 nerve growth factor receptors. CSA bound nine proteins, including ROBO2, 2 EPHs, 1 EFN, two SEMAs, and netrin 4. HA bound no ECM nerve-related epitopes. SPR confirmed that KS binds SLIT2 strongly. The KS core protein mimecan/osteoglycin bound 15 proteins.


CONCLUSIONS
Corneal stromal GAGs bind, and thus could alter the availability or conformation of, many proteins that may influence keratocyte and nerve growth cone behavior in the cornea.
Conrad
Abigail H
AH
Division of Biology, Kansas State University, Manhattan, Kansas 66506-4901, USA. aconrad@ksu.edu
Zhang
Yuntao
Y
Tasheva
Elena S
ES
Conrad
Gary W
GW
D000818:Animals; D019921:Biotinylation; D002417:Cattle; D002809:Chondroitin Sulfates / Q000378:metabolism*; D003315:Cornea / Q000166:cytology / Q000294:innervation / Q000378:metabolism*; D000939:Epitopes / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005399:Fishes; D020439:Growth Cones / Q000502:physiology; D006820:Hyaluronic Acid / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D007632:Keratan Sulfate / Q000378:metabolism*; D015603:Keratinocytes / Q000378:metabolism; D009412:Nerve Fibers / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009672:Notochord / Q000166:cytology / Q000294:innervation / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D040901:Proteomics / Q000379:methods*; D020349:Surface Plasmon Resonance
D000939:Epitopes; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates; D007632:Keratan Sulfate