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Macular Corneal Dystrophy: Failure to Synthesize a Mature Keratan Sulfate Proteoglycan

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Macular Corneal Dystrophy: Failure to Synthesize a Mature Keratan Sulfate Proteoglycan Proc. Natl. Acad. Sci. USA Vol. 77, No. 6, pp. 3705-3709, June 1980 Medical Sciences Macular corneal dystrophy: Failure to synthesize a mature keratan sulfate proteoglycan (human genetic disease/glycoprotein processing/blindness/extracellular matrix/corneal stroma) JOHN R. HASSELL*, DAVID A. NEWSOME*, JAY H. KRACHMERt, AND MERLYN M. RODRIGUESt *Section on Retinal and Ocular Connective Tissue Diseases, Clinical Branch, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20205; tDepartment of Ophthalmology, University of Iowa, Iowa City, Iowa 52242; and tSection on Ocular Pathology, Clinical Branch, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20205 Communicated by Elizabeth F. Neufeld, March 20,1980 ABSTRACT Corneal specimens obtained during surgery dystrophy is first detected before age 10 yr. Visual acuity pro- from patients with macular corneal dystrophy and obtained at gressively deteriorates until corneal transplantation is required, autopsy from control eyes were incubated in a medium con- usually by age 25-30 yr. The small amount of tissue produced taining radioactive precursors of glycoproteins and proteogly- cans. Biosyntheticallyradiolabeled material was extracted and by transplantation is insufficient for direct chemical analysis. characterized by using molecular sieve chromatography and Furthermore, stromacytes derived from normal corneas and specific enzymes. Cells in control corneas synthesized both a grown in cell culture do not synthesize the proteoglycans chondroitin sulfate proteoglycan and a keratan sulfate proteo- characteristic of the stroma (10). Therefore, the organ culture glycan similar to those present in monkey and bovine corneas. system appeared to be a promising technique for studying the Cells in macular corneas synthesized a normal chondroitin proteoglycans of macular corneas obtained from keratoplasty. sulfate proteoglycan but did not synthesize either keratan sul- fate or a mature keratan sulfate proteoglycan. Instead, macular The data presented here suggest that the cloudiness in corneas corneas synthesized a glycoprotein with unusually large oligo- from patients with macular corneal dystrophy results from the saccharide side chains. This glycoprotein was not detected in failure to synthesize a mature keratan sulfate proteoglycan. normal corneas and is slightly smaller than normal keratan sulfate prot can. The failure to synthesize a mature keratan MATERIALS AND METHODS sulfate proteoglycan may produce corneal opacity and result in blindness. Because of evidence indicating that the corneal Organ Culture Procedures. Corneas excised with a 7- to keratan sulfate proteoglycan is normally synthesized through 8-mm trephine blade were obtained from human eyes at ker- a glycoprotein intermediate [Hart, G. W. & Lennarz, W. (1978) atoplasty or from control donor eyes at autopsy. Corneas ob- J. Biol. Chem. 253,5795-5801, macular corneal dystrophy may tained at surgery included three from two patients with macular be a defect in glycoprotein processing. dystrophy (here termed "macular corneas"), one from a patient Macular, lattice, and granular corneal dystrophies are charac- with lattice dystrophy, two from two patients with granular terized by specific abnormalities of the corneal stroma and dystrophy, one from a patient with keratoconus, and two from result in loss of vision (1). Macular corneal dystrophy is char- normal donor eyes (here termed "control corneas"). Specimens acterized clinically by recessive inheritance, a general diffuse were placed immediately in Eagle's minimal essential medium haze throughout the corneal stroma, and the accumulation of supplemented with 5% fetal calf serum, 2 mM glutamine, and irregularly shaped deposits which tend to be anterior in the 100 units of penicillin and 50 ,ug of streptomycin per ml. The central region of the stroma. Histologically, the deposits are medium also contained 200-400 ,Ci of [6-3H]glucosamine per primarily extracellular and stain positively with Alcian blue and ml and 1.0-1.5 mCi of Na235SO4 per ml (New England Nuclear; the periodic acid-Schiff reagent (2, 3). These observations 1 Ci = 3.7 X 1010 becquerels). The corneas were incubated at suggest that the deposits may be glycosaminoglycans and oli- 370C for 18-20 hr in 5% C02/95% air at 100% humidity. At gosaccharides accumulating in the stromal extracellular ma- the end of the incubation period the corneas were rinsed briefly trix. in phosphate-buffered saline and stored frozen at -700C. The extracellular matrix of the normal corneal stroma consists Biochemical Procedures. Each cornea was individually primarily of type I collagen and two proteoglycans: a chon- thawed, the epithelium and endothelium were removed rapidly droitin-dermatan sulfate proteoglycan and a keratan sulfate by scraping, and the stroma was placed in 1.0 ml of extraction proteoglycan (4-9). Because most of the sulfated carbohydrate solvent containing 4 M guanidine-HCL 0.1 M 6-aminohexanoic in the cornea is contained in these two proteoglycans, macular acid, 0.01 M Na2 EDTA, 5 mM benzamidine hydrochloride, corneal dystrophy may involve alterations in one or both of and 0.5 M sodium acetate (pH 5.8-6.0) for 24 hr at 4°C (11). these proteoglycans. Recently, an organ culture system utilizing The extracting solvent was removed, and the stroma was monkey corneas has been developed that allows the biosynthesis reextracted in 0.5 ml of the same solvent for 4-6 hr at 40C. The of radiolabeled corneal stromal proteoglycans in vItro (9). This extracts were combined, and unincorporated radioactivity was system involves incubating intact corneas in a tissue culture removed by chromatography on a Sephadex G-25 PD-10 col- medium containing radioactive precursors. Analyses of the umn (Pharmacia) that had been equilibrated with and eluted radiolabeled proteoglycans synthesized during organ culture with 4 M guanidine-HCI/0.02 M Tris-HCI, pH 7.0. The residual indicated that they were representative of the proteoglycans tissue was digested with 0.5 mg papain (Sigma) in 1.0 ml of 1 that accumulate in the stromal extracellular matrix. M sodium acetate, pH 6.5/5 mM cysteine/5 mM EDTA at The corneal cloudiness in individuals with macular corneal 55-i60C for 6 hr. Any unincorporated radioactivity in the di- gest was removed by chromatography on PD-10 columns as The publication costs of this article were defrayed in part by page described above. charge payment. This article must therefore be hereby marked "ad- The glycoconjugates in the 4 M guanidine.HCI extract were verttsement" in accordance with 18 U. S. C. §1734 solely to indicate fractionated on a Sepharose CL-4B column (200 X 1.6 cm; 4.2 this fact. ml collected per tube), equilibrated, and eluted with 4 M gua- 3705 Downloaded by guest on September 25, 2021 3706 Medical Sciences: Hassell et al. Proc. Natl. Acad. Sci. USA 77 (1980) nidine-HCI/0.02 M Tris, pH 7.0. The radioactively labeled peak fractions were pooled, dialyzed against distilled water, and 250C lyophilized. These peak fractions were then dissolved in 4 M guanidine-HCI/0.02 M Tris, pH 7.0, and divided into two parts. 200C 400 One part was directly applied to a Sepharose CL-6B column (90 X 1.5 cm, 2.0 ml collected per tube), equilibrated, and 150C 300 eluted with 4 M guanidine.HCl/0.02 M Tris.HCl, pH 7.0. The 200 second part was digested with papain and fractionated on 6-00 Sepharose CL-6B as described to isolate the glycosaminoglycans 50C and glycopeptides. The tubes containing the peak fractions 1- F. UE were pooled, dialyzed against distilled water, and lyophi- 8000 0 lized. :r: 10,000 1250 >6 The glycosaminoglycans and glycopeptides isolated from Sepharose CL-6B were sequentially digested with chondroiti- 8000 1000 nase ABC (Miles), keratanase (keratan sulfate-f3-endogalac- tosidase, a gift from Sakaru Suzuki), and nitrous acid as de- 6000 750 scribed (9, 12). In brief, the samples were digested with chon- 4000 500 droitinase ABC and chromatographed on Sephadex G-50 (0.6 X 30cm, 1.0 ml collected per tube). The amount digested was 2000 250 determined by the shift in elution position from a higher to a lower molecular weight. The undigested higher molecular 10 20 30 40 50 60 70 80 weight material was then digested with the keratanase and the 9V amount digested was determined again. Undigested samples Vo eluted from the same column served as "blanks." Samples also Tube number were digested for 18 hr at 370C with 2 units of mixed exogly- FIG. 1. Sepharose CL-4B chromatography of 4 M guanidine.HCl cosidases per ml (Miles) in 0.4 M sodium acetate buffer, pH extracts of (A) control and (B) macular corneal stromas. Corneas were 4.4/0.1 M NaCI. radiolabeled in organ culture with [3H]glucosamine and Na235SO4 Radioactivities in fractions were measured by liquid scin- before extraction. Brackets denote pooled tubes. tillation spectrophotometry with 10 ml of ACS (Amersham). Aliquots (0.01-0.2 ml) from each tube were assayed for radio- of this material. Similar results were obtained by chromatog- activity. An equivalent amount of 70% ethanol was added when raphy of intact (Fig. 2C) and degraded (Fig. 2D) peak 2 ma- measuring samples in 4 M guanidine.HG. An external standard terial from macular corneas on Sepharose CL-6B. was used to correct for spillover of M5S into the 3H channel. Peak 3 material, isolated by Sepharose CL-4B chromatog- raphy, was chromatographed on Sepharose CL-6B. Intact peak RESULTS 3 material from control corneas (Fig. 3A) eluted as a single peak There were two major differences between labeled macro- at tube 30. After papain digestion, two radiolabeled peaks were molecules obtained from control and macular corneas incu- observed (Fig. 3B): a glycosaminoglycan peak at tube 41 bated with [3H]glucoaaniine and [35Sisulfate. First, substantially (designated peak 3b) with a preceding shoulder (designated less of the total incorporated 3H and [35S]sulfate in macular peak 3a) containing both 3H and 35S and another peak at tube corneas was extracted by 4 M guanidine.HCl.
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