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Characterization of a novel family of ciliary body glycoproteins.

Publication ,  Journal Article
Howell, DN; Ross, JR; Miller, SE; Sanfilippo, FP
Published in: Invest Ophthalmol Vis Sci
July 1994

PURPOSE: To isolate and characterize ciliary body epithelial antigens reactive with a monoclonal antibody, 2B4.14.1. METHODS: A mouse monoclonal antibody generated against human corneal endothelium, 2B4.14.1 reacts with nonpigmented epithelium of human and guinea pig ciliary bodies. The ciliary body proteins reactive with 2B4.14.1 were identified by Western blotting and were partially purified by affinity chromatography with 2B4.14.1 coupled to a solid support matrix. Carbohydrate components of the antigenic molecules were analyzed by lectin chromatography and by digestion with the enzymes N-glycosidase F and endoglycosidase F. The cellular and subcellular distribution of the antigens was examined by immunoperoxidase staining and by immunoelectron microscopy of ultracryotome sections of ciliary body. RESULTS: 2B4.14.1 reacted with families of guinea pig and human ciliary body glycoproteins with estimated molecular weights ranging from 250 to 325 kD. In Western blots of samples reduced before electrophoresis, the high molecular weight bands were replaced by weakly reactive bands at 115 to 130 and 210 kD, indicating that the 2B4.14.1 ligands have disulfide bonds. 2B4.14.1 ligands from both guinea pig and human ciliary body were bound by immobilized lectins, including concanavalin A, Datura stramonium lectin, and Lens culinaris hemagglutinin, which recognize components of N-linked oligosaccharides. Guinea pig ciliary body antigens digested with N-glycosidase F and endoglycosidase F failed to react with 2B4.14.1 in Western blots, confirming the presence of N-linked oligosaccharide chains and indicating that they form an integral part of the 2B4.14.1-reactive antigenic site. Molecular weight shifts of glycosidase-digested antigens were consistent with the presence of two to four N-linked oligosaccharide units. In immunoperoxidase-stained sections of guinea pig and human ciliary body, 2B4.14.1 reacted primarily with nonpigmented epithelial cells. Staining of guinea pig epithelial cells was fairly uniform; staining of human epithelial cells was concentrated on the basal surface. By immunoelectron microscopy, a majority of the 2B4.14.1 antigenic reactivity was localized immediately external to the nonpigmented epithelial cell plasma membrane. CONCLUSIONS: 2B4.14.1 reacts with a novel family of high molecular weight glycoproteins associated with the nonpigmented epithelial cell surface in guinea pig and human ciliary body.

Duke Scholars

Published In

Invest Ophthalmol Vis Sci

ISSN

0146-0404

Publication Date

July 1994

Volume

35

Issue

8

Start / End Page

3308 / 3318

Location

United States

Related Subject Headings

  • Pigment Epithelium of Eye
  • Ophthalmology & Optometry
  • Oligosaccharides
  • Middle Aged
  • Microscopy, Immunoelectron
  • Membrane Glycoproteins
  • Immunoenzyme Techniques
  • Humans
  • Guinea Pigs
  • Ciliary Body
 

Citation

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MLA
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Howell, D. N., Ross, J. R., Miller, S. E., & Sanfilippo, F. P. (1994). Characterization of a novel family of ciliary body glycoproteins. Invest Ophthalmol Vis Sci, 35(8), 3308–3318.
Howell, D. N., J. R. Ross, S. E. Miller, and F. P. Sanfilippo. “Characterization of a novel family of ciliary body glycoproteins.Invest Ophthalmol Vis Sci 35, no. 8 (July 1994): 3308–18.
Howell DN, Ross JR, Miller SE, Sanfilippo FP. Characterization of a novel family of ciliary body glycoproteins. Invest Ophthalmol Vis Sci. 1994 Jul;35(8):3308–18.
Howell, D. N., et al. “Characterization of a novel family of ciliary body glycoproteins.Invest Ophthalmol Vis Sci, vol. 35, no. 8, July 1994, pp. 3308–18.
Howell DN, Ross JR, Miller SE, Sanfilippo FP. Characterization of a novel family of ciliary body glycoproteins. Invest Ophthalmol Vis Sci. 1994 Jul;35(8):3308–3318.

Published In

Invest Ophthalmol Vis Sci

ISSN

0146-0404

Publication Date

July 1994

Volume

35

Issue

8

Start / End Page

3308 / 3318

Location

United States

Related Subject Headings

  • Pigment Epithelium of Eye
  • Ophthalmology & Optometry
  • Oligosaccharides
  • Middle Aged
  • Microscopy, Immunoelectron
  • Membrane Glycoproteins
  • Immunoenzyme Techniques
  • Humans
  • Guinea Pigs
  • Ciliary Body