1989, Protein-Related Abnormalities in Keratoconus

8/17/2019 1989, Protein-Related Abnormalities in Keratoconus

http://slidepdf.com/reader/full/1989-protein-related-abnormalities-in-keratoconus 1/7

Investigative Ophthalmolog y & V isual Science, Vol. 30, No . 12, Decem ber 1989

Copyright © Association for Research in Vision and Ophthalmology

Protein-Related Abnormalities in Keratoconus

Noorjahan Panjwani,*j- Jim Drysdale,t Brian Clark,* John Alberta,f and Jules Baum*}:

Two-dimensional electrophoretic maps of extracts from eleven normal and eleven keratoconus corneas

were compared. Of the eleven corneas analyzed, eight were pooled and the remaining three were

analyzed individually. Several differences were demonstrated between electrophoretic patterns of

normal and keratoconus corneas. In keratoconus corneas, 1) two abnormal compon ents M W 54 kD

and 26kD) were observed; 2) three normal corneal components M W 12kD , 14k D, and 39k D) were

present in significantly h igher amounts; and 3) three normal corneal proteins M W 6 6k D , 55k D, and

13kD) were present in reduced amounts. The molecular weight and isoelectric point of one of the

normal corneal proteins that we found to be reduced in keratoconus corneas were close to that of a

subunit of prolyl-4-hydroxylase, an enzyme required for hydroxylation of proline residues of collagen.

The possibility the abnormal proteins detected in the keratoconus corneas were derived from those

normal corneal proteins which were absent or were present in reduced amounts in the keratoconus

corneas remains to be established. This study may provide protein markers for elucidation of the

biochemical abnormality in keratoconus. Invest Ophthalmol Vis Sci 30:2481-2487,1989

Keratoc onus is characterized by thinnin g and scar-

ring of the central cornea. It is slowly progressive and

either bi- or unilateral. Although the clinical and ul-

trastructural characteristics of the disease are well es-

tablished,

1

3

the biochemical defect in keratoconus

has not been elucidated. H istochemical as well as bio-

chemical approaches have not detected significant

differences in collagen types between normal and

keratoconus corneas.

4

6

It has been shown that media

incubated with keratoconus corneas, as well as stro-

mal cells in culture derived from keratoconus cor-

neas,

contain higher levels of collagenolytic activity

compared to normal controls,

7

9

a result which sug-

gests a decrease in corneal collagen content in kera-

toconus. Attempts to demonstrate reduced collagen

levels in keratoconus corneas, however, have pro-

vided conflicting results.

5

'

7

'

9

13

Cannon and Foster

14

reported abnormally high levels of lysinorleucine

crosslinks of collagen in keratoconus; these findings,

however, could not be confirmed.

15

Biochemical ap-

proaches to detect differences related to proteogly-

cans and glycosaminoglycans have also provided

variable data. Hassell et al

16

reported that the pattern

From the Departments of Ophthalmology and fBiochemistry,

Tufts University School of Medicine, and ^New England Medical

Center, B oston, Massachusetts.

Supported by the National Institutes of Health Grants

EY-07088, EY-04034, and DDK-32455.

Submitted for publication: August 1, 1988; accepted May 12,

1989.

Reprint requests: Noorjahan Panjwani, PhD, Department of

Ophthalmology, Tufts University School of M edicine, 136 Harri-

son Avenue, Box 450, Boston, MA 02111.

of proteoglycans synthesized by keratoconus corneas

in organ culture was indistinguishable from that syn-

thesized by norm al corn eas. In an earlier study, it was

reported that the hexosamine concentration in kera-

toconus corneas was about 40% greater than nor-

mal.

10

Anseth,

17

however, found that the concentra-

tion of hexosamine was identical in keratoconus and

normal corneas. Andreassen et al

12

found no differ-

ences in uronic acid concentration in normal and

keratoconus corneas; in contrast, in a recent histo-

chemical study, an abnormally high amount of poly-

anions that include primarily glycosaminoglycans

was found in keratoconus corneas when compared to

normal controls.

18

In another study, using a mono-

clonal antibody specific for keratan sulfate, it was

shown that compared to normal controls, some kera-

toconus corneas contained less keratan sulfate.

19

Thus,

in spite of a num ber of well-designed studies,

the exact biochemical abnormality responsible for

the development of keratoconus has yet to be eluci-

dated. A major obstacle in the study of keratoconus

has been the unavailability of specific biochemical

markers with which investigations can be designed.

In various nonocular studies, comparison of two-di-

mensional gel electrophoretic patterns of normal and

diseased tissue have led to the identification of the

disease-related proteins.

20

'

21

Two-dimensional elec-

trophoresis is a powerful research tool pe rmitting res-

olution of up to 1,000 proteins from a single human

cell type.

22

To our knowledge, two-dimensional elec-

trophoretic patterns of the complete extracts of nor-

mal or keratoconus corneas have not been compared

to date. The purpose of this study is to compare two-

2481

ownloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/Journals/IOVS/933145/ on 05/04/2016



2 4 8 2

INVESTIGATIVE OPHT HALM OLOG Y VISUAL SCIENCE / Dec em be r 19 89

Vol. 30

dimensional maps of extracts of normal and kerato-

conus corneas and thereby identify protein markers

that may be specific to the disease.

Materials and Methods

Tissue

Human keratoconus buttons were obtained at the

time of keratoplasty. With the exception of the kera-

toconus, the patients undergoing keratoplasty were

clinically normal; there was no evidence of general-

ized connective tissue disorders. The diseased corneas

were rinsed three times with saline and kept frozen at

-80°C or in liquid N

2

until used. Normal central

human corneas were excised with an 8-mm trephine

from frozen eyes supplied by the Lions Eye Bank

(Seattle, WA) an d were rinsed three times with saline

prior to use. For the normal eyes, the average time

period between death and enucleation was approxi-

mately 7 hr, and between enucleation and freezing,

approximately 6 hr. Eleven normal corneas and

eleven keratoconus buttons were used. Within each

group, eight corneas were pooled and the remaining

three were investigated individually. Ages of the nor-

mal corneal donors and of the patients with kerato-

conus are shown in Table 1. Information concerning

the extent of scarring in keratoconus buttons is also

shown in Table 1.

Extraction of Proteins from Normal and

Keratoconus Corneas

The epithelium was scraped off the corneas while

they were still frozen. Th e corneas were then minced

into six to eight pieces with a scalpel, on dry ice. The

minced tissue was thawed directly in ice-cold 4 M

guanidine hydrochloride (1-1.5 ml/cornea), and ex-

traction was carried out for 16-18 hr on a wrist action

shaker in the presence of a solution of protease inhibi-

tors,

composed of raM phenylmethylsulfonylfluor-

ide,

1 mM benzamidine hydrochloride, 100 mM 6-

aminohexanoic acid, and 10 mM N-ethylmaleimide.

The extract was collected by decantation, and the

residue was reextracted as described above for an ad-

ditional 6-8 hr. Both supernatants were pooled, dia-

lyzed extensively against cold water, and lyophilized.

The lyophilized material was suspended in cold dis-

tilled water and aliquots were used for protein deter-

mination and two-dimensional gel electrophoresis.

Protein Determination

The protein concentration in the corneal extracts

was determined by the bicinchoninic acid (BCA)

method

23

(Pierce Chemical, Rockford, IL) using bo-

vine serum albumin as a standard. Protein yield in

the extract ranged from 400-650 /Ltg/comea.

Two Dimensional Gel Electrophoresis

Two-dimensional electrophoresis was performed

according to the method of O'Farrell

22

by Kendrick

Laboratories (Madison, WI) as follows. Isoelectric fo-

cusing using ampholines at 1.5%, pH 5-7; 1.5%, pH

5-8; and 1.0%, pH 3.5-10 (LKB Instruments, Balti-

more, MD) was carried out in 135-mm X 2-mm

tubes at 400 V for 12 hr followed by 800 V for 30

min. Forty ng of an isoelectric focusing internal stan-

dard, vitamin D-dependent calcium-binding protein,

MW 27,000 and pi 5.2, was added to the samples.

The final tube gel pH gradient was estimated by a

surface pH electrode (Biorad, Richmond, CA) and by

colored acetylated cytochrome pi markers (Calbio-

chem-Behring, La Jolla, CA) run in an adjacent tube.

Electrophoresis in the second dimension was per-

formed in 135-mm X 150-mm X 0.75-mm slab gels

containing 10% acrylamide. The following proteins

(Sigma Chemical, St. Louis, MO) were added as mo-

lecular weight standards to the agarose that sealed the

tube gels to the slab gels: myosin heavy chain (220

kD),

phosphorylase A (94 kD), catalase (60 kD), actin

(43 kD) and lysozyme (14 kD). Protein spots were

visualized by silver staining according to the proce-

dure of Oakley et al.

24

Table 1. Ages of normal corneal donors and of patients with keratoconus at the time of keratoplasty

Group

number

1

2

3

4

5

6

7

8

Cornea

normal

no rma l

normal

no rma l

keratoconus

keratoconus

keratoconus

keratoconus

Number of

corneas

8

ii

Age (yr)

28-57 (mean: 41)

47

47

47

26-41 (mean: 33)

47

52

40

Degree of

scarring

_

-

-

-

- t o + +

+

+ + +

+

of button

scarred

_

-

-

-

0-10

<5

15

<5

Scarring: — none, + mimimal, ++ moderate, +++ severe.




No .

12

PROTEIN-RELATED ABNORMALITIES IN KERATOCONUS / Pa nj w on i er a l

2 4 8 3

Two-dimensional electrophoretic maps of normal

corneas were compared with each other and with

those of keratoco nus corneas by overlapping the gels

on a light box. The spots which were present only in

the diseased corneas, or which were present in the

diseased corneas in higher concentrations, were desig-

Fig. 1. Two-dimensional

electrophorograms of ex-

tracts prepared from eight

pooled normal (N) and

eight pooled keratoconus

(K) corneas. Ninety micro-

grams of protein was ap-

plied on each isoelectric

focusing (IEF) gel, and

polypeptide spots were vi-

sualized by silver staining.

The basic side is on the

right, the acidic on the left;

the high molecular weight

region is at the top. The

final tube gel pH gradient

was estimated by a surface

pH electrode and by colored

acetylated cytochrome pi

markers run in an adjacent

tube. Electrophoresis (SDS)

in the second dimension

was performed in 10% ac-

rylamide slab gels. Myosin

heavy chain (220 kD),

phosphorylase A (94 kD),

catalase (60 kD), actin (43

kD) and lysozyme (14 kD)

were added as molecular

weight standards to the aga-

rose which sealed the tube

gels to the slab gels. These

standards appear as fine

horizontal lines. In addi-

tion, an IEF internal stan-

dard, vitamin D-dependent

calcium binding protein,

MW 27 kD and pi 5.2, was

added to the samples. This

standard is indicated by an

arrowhead in each gel. The

spots which were present

only in diseased corneas, or

which were present in the

diseased corneas in higher

concentrations, were desig-

nated as K spots; the spots

which were present in lower

intensity in the diseased

corneas as compared to

normal corneas were desig-

nated as N spots. Arrows

point to various N and K

spots or to their approxi-

mate positions when the

spots were absent in either

gel.

1 4 -

8 0




2 4 8 4

INVESTIGATIVE OPHTHALM OLOG Y G VISUAL SCIENCE / De ce m be r 19 89

Vol. 30

CO

LU

O

<

GO

<

CD

2 3

NORMAL

5 6 7 8

KERATOCONUS

Fig.

2. Comparison of intensities of K spots (see Fig. 1) in all

normal and diseased corneas analyzed in this study. Electropho-

retic patterns no. 1 and no. 5 were obtained from extracts of eight

pooled normal and eight pooled diseased corneas, respectively; the

nated as K spots. The spots which were absent, or

which were present in lower intensity, in the diseased

corneas as compared to normal corneas were desig-

nated as N spots. Approximate intensities of the N

and K spots were measured using an Ultroscan XL

laser densitometer (LKB). The polypeptide densities

in the respective gels were normalized against two

reference proteins which had constant density ratios

to each other in all gels. The densities of these two

proteins in each gel were used to create a normaliza-

tion factor for each gel compared to an arbitrarily

selected standard gel.

Results

Comparison of two-dimensional maps of normal

and k eratoconus corneas revealed that

1)

two compo-

nents,

Kl and K5, were detected in keratoconus cor-

neas, but not in normal corneas (Figs. 1, 2); 2) three

components, K2-K4, were present in significantly

higher amounts in keratoconus corneas compared to

those in normal corneas (Figs. 1, 2); and 3) three

normal corneal proteins, N1-N3, were present in sig-

nificantly lower amounts in keratoconus corneas

(Figs. 1, 3). Molecular weights and isoelectric points

of

all

N and K proteins are shown in Table 2. Inten-

sities of various K and N spots are shown in Figures 2

and 3, respectively. As shown in Figure 1, N2 con-

sisted of several spots. For density measurement of

N2,

the area representing several spots was analyzed

as a whole, and the intensity of the various spots

within the N2 area was not measured individually.

The density of component N3 was not measured be-

cause in a number of gels this component was not

resolved from lysozyme, which was used as an inter-

nal standard

Fig. 1).

Difference in the intensity of N3

between normal and keratoconus corneas was more

prominent in the two-dimensional maps of individ-

ual corneas (not shown) than in those of the pooled

corneas (Fig. 1). The broad band migrating beneath

the K3 position in the two-dimensional patterns of

normal corneas comigrated with K3 in the two-di-

mensional patterns of pooled as well as two of the

three individual keratoconus corneas.

A mixed extract of keratoconus and normal cor-

neas was also analyzed to determ ine whether those N

and K components w hich were found to be present in

both normal and keratoconus comigrate. As shown

in Figure 4, components N1-N3 and K2-K4, which

remaining electrophoretic patterns were obtained from individual

corneas. Ninety m icrograms of protein was loaded on each gel, and

polypeptide densities in the respective gels were normalized against

two reference proteins as described in the text.




No. 12

PROTEIN-RELATED ABNORMALITIES IN KERATOCONUS / Po nj w on i er ol

2 4 8 5

CO

LU

O

z

<

m

O

O

3

2

1

NORMAL

KERATOCONUS

Fig. 3. Comparison of intensities of N spots (see Fig. 1) in all

normal and diseased corneas analyzed in this study. Electropho-

retic patterns no. and no. 5 were obtained from extracts of eight

pooled normal and eight pooled diseased corneas, respectively; the

remaining electrophoretic patterns were obtained from individual

corneas. Ninety micrograms of protein was loaded on each gel, and

polypeptide densities in the respective gels were normalized against

two reference proteins as described in the text.

were present in both keratoconus and normal cor-

neas, though in varying amounts, comigrate.

Among all the keratoconus corneas we studied, the

data derived from case no . 6 appeared to deviate sig-

nificantly from those of the rest of the diseased cor-

neas.

As shown in Figure 4, in the case-6 cornea,

components Kl, K2, and K5 were present only in

trace amounts. In contrast, these components were

present in substantially greater amounts in all other

keratoconus corneas studied. The cornea of case 6

was also dissimilar from the other keratoconus cor-

neas studied in that it contained a higher amount of

component

N1,

compared to all other diseased speci-

mens (Fig. 3).

Discussion

We have demonstrated that in keratoconus corneas

several abnormal proteins are present and that the

amounts of several normal corneal proteins are re-

duced. Specifically, our data indicate that in kerato-

conus corneas, two abnormal components (Kl and

K5) are present and three normal corneal compo-

nents (K2-K4) are present in significantly higher

amounts. In addition, three normal corneal proteins

(Nl ,

N2, and N3) are present in reduced amounts in

the diseased co rneas.

The molecular weight and isoelectric point of the

normal corneal component Nl, detected only in

trace amounts in keratoconus corneas, is close to tha t

of a subunit of prolyl-4-hydroxylase.

25

It remains to

be determined whether component N is a subunit of

prolyl hydroxylase, but one study has suggested an

elevated level of prolyl-4-hydroxylase activity in ker-

atoconus corneas.

9

Repor ts in the literature regarding

hydroxyproline concentration in keratoconus con-

flict; some have reported reduced levels,

9

whereas

others have found identical concentrations of this

imino acid in normal and keratoconus corneas.

71 2

It is possible that the abnormal proteins we de-

tected in keratoconus corneas may have been derived

from those normal corneal proteins which appear to

be absent, or present in reduced amounts, in kerato-

conus corneas. These abnorm al pro teins may have an

altered or truncated amino acid sequence, or they

may be proteins with a normal amino acid sequence

that may have undergone abnormal processing, such

as insufficient or excessive glycosylation, sulphation,

or phosphorylation. For example, abnormal protein

K2 (MW 12kD, pi 5.35) could have been derived by

alteration of the amino acid sequence of the normal

corneal protein N3 (MW 12kD, pi 5.7), resulting in

the decrease of the isoelectric po int. It is unlikely that

the abnormal K proteins are degradation products of

N p roteins, because all extractions were performed at

low temperatures in the presence of various protease

inhibitors. How ever, because the average time period

between enucleation and freezing of the normal eyes

was about 7 hr, it is possible, although unlikely, that

K spots were detected in lesser amounts in normal

corneas as a result of proteolytic degradation.

It remains to be determined whether the abnorm al

corneal proteins we detected in keratoconus corneas

are involved in the development of the disease. It is

Table 2.

Molecular weights and isoelectric p oints

of various N and K components

Component

K l

K 2

K 3

K 4

K 5

N l

N 2

N 3

Molecular

w i ht

(kD)

54

12

39

15

28

62-72

52-60

13

Isoelectric

point (pi)

5.25

5.35

6.45

7.38

6.75

5.20 (4.9-5.3)

5.5-5.95

5.7




2 4 6 6

INVESTIGATIVE OPHTHALMOLOGY 6 VI5UAL 5CIENCE D e c e m b e r 1989

Vol. 30

1 4 -

Fig.

4. A

two-dimen-

sional electrophorogram

of

a mixed extract of normal

and keratoconus corneas.

Equal amounts

of

kerato-

conus and normal corneal

extracts were mixed,

and a

total of 90 n protein was

applied on the isoelectric

focusing gel.

For

other

ex-

perimental details, see leg-

end for Figure I. Note that

all

N and K

components,

which

are

present

in

both

normal and keratoconus

corneas, com igrate

in the

mixed extract. IEF = iso-

electric focusing; SDS

= SDS polyacrylamide

gel

elect rophores is.

ao

possible that the abnormal electrophoretic pattern we

found in keratoc onus cor neas is at least in part due to

the nonspecific scarring associated with keratoconus.

This study may provide marker proteins for the elu-

cidation of the biochemical abnormality in kerato-

conus.

Key words: keratoconus two-dimensional gel electrophore-

sis, proteins cornea.

Acknowledgments

We thank Drs. Juan J. Arentsen Ann M. Bajart S.

Arthur Boruchoff Helen Boisjoly Elisabeth J. Cohen Jay

Krachmer Peter R. Laibson Roger F. Meyer H. Kaz

Soong Alan Sugar and Roger F. Steinert for providing

keratoconus specimens.

References

1. Chi HH, Katzin HM, and Teng

CC:

Histopathology of kerato-

conus. Am

J

Ophthalmol 42:847, 1981.

2. Teng CC: Electron microscopic study

of

the pathology

of

the

keratoconus:

I.

Am

J


3. Pataa CL, Joyon L,

and

Roucher E: Ultrastructure

of

kerato-

conus. Arch Ophthalmol (Paris) 30:403,

1970.

4.

Newsome DA, Foidart JM , Hassell JR, K rachmer JH, Rodri-

gues MM,

and

Katz

SI:

Detection of specific collagen types in

normal

and

keratoconus corneas. Invest Ophthalmol Vis

Sci

20:738, 1981.

5. Radda TM, Menzel EJ Freyler H and Gnad HD : Collagen

types in keratoconus. Graefes Arch Klin Exp Ophthalmol

218:262, 1982.

6. Nakayasu

K

Tanaka

M

Konomi

H and

Hayashi

T:

Distri-

bution

of

types

I II III IV and V

collagen

in

normal

and

keratoconus corneas. Ophthalmic Res 18:1,

1986.

7. Kao WW, Vergens J, Ebert J, Sundar Raj CV, and Brown ST:

Increased collagenase and gelatinase activities

in

keratoconus.

Biochem Biophys Res Commun 107:929, 1983.

8. Rehany U, Lahav M, and Shoshan S: Collagenolytic activity in

keratoconus. Ann Ophthalmol 14:751, 1982.

9. Ihatainen A Salo T Forsius H and Peltonen L: Increase in

type I and type IV collagenolytic activity in primary cultures of

keratoconus cornea. Eur J Clin Invest 16:78, 1986.

10.

Buddecke E and W otlensak J: Saure mucopolysaccharide und

glykoproteine

der

menschlichen cornea

in

abhangigkeit

vom

lebensalter und bei keratoconus. Graefes Arch Klin Exp


11.

Robert L, Schillinger G, Moczar M, Junq ua S, and Moczar E:

Biochemical studies of keratoconus. Arch Ophthalmol (Paris)

30-590, 1970.

12. Andreassen TT, Simonsen AH, and Oxlund H: Biochemical

properties of keratoconus and normal corneas. Exp Eye Res

31:435. 1980.

13.

Yue BYJT, Sugar J, and Benveniste K: Heterogeneity in kera-

toconus: Possible biochemical basis. Proc Soc Exp Biol Med

175:336, 1984.

14.

Cannon

DJ and

Foster CS: Collagen crosslinking

in

kerato-

conus. Invest Ophthalmol Vis Sci 17:63, 1978.

15. Oxlund H and Simonsen AH: Biochemical studies of normal




No . 12 PROTEIN-RELATED ABNORMALITIES IN KERATOCONUS / Pa nj w an i er al 2 4 8 7

and keratoconus corneas. Acta Ophthalmol (Copenh) 63:666,

1985.

16. Hassell JR, Newsome DA, Krachmer JH , and Rodrigues MM:

Macular corneal dystrophy: Failure to synthesize a mature

keratan sulfate proteoglycan. Proc Natl Acad Sci USA

77:3705, 1980.

17.

Anseth A: Studies on corneal polysaccharides. Exp Eye Res

8:438, 1969.

18. Yue BY JT, Sugar J, and Schrode K: Histochemical studies of

keratoconus. Curr Eye Res 7:81, 1988.

19.

Conrad GW, Funderburgh ML, Funderburgh JL, and Rodri-

gues MM: Keratan sulfate in keratoconus, pellucid degenera-

tion, and macular corneal dystrophy. ARVO Abstracts. Invest

Ophthalmo l Vis Sci 29(Suppl):215, 1988.

20. Harrington M G, Merril CR, Goldman D, Xu X, and McFarlin

DE:

Two-dimensional electrophoresis of cerebrospinal fluid

proteins in multiple sclerosis and various neurological diseases.

Electrophoresis 5:236, 1984.

21. Jellum E and Tho rsrud A K: Detection of cancer-related pro-

teins by two-dimensional electrophoresis. Ann NY Acad Sci

428:173,

1984.

22.

O'Farrell PH: High resolution two-dimensional electrophore-

sis of proteins. J Biol Chem 250:4007, 1975.

23. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner

FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ,

and Klenk DC: Measurement of protein using bicinchoninic

acid. Anal Biochem 150:76, 1985.

24.

Oakley BR, Kirsch DR, and Morris NR: A simplified ultra-

sensitive silver stain for detecting proteins in polyacrylamide

gels. Anal Biochem 105:361, 1980.

25. Berg RA, Kedersha NL, and Guzman NA: Purification and

partial characterization of the two nonidentical subunits of

prolyl hydroxylase. J Biol Chem 254:3111, 1979.

Documents

1989, Protein-Related Abnormalities in Keratoconus