Variability of keratan sulfate isolated from beef cornea

28~

BIOCHIMICA F.T BIOPHYSICA AC'I'A

VARIABILITY OF KERATAN SULFATE ISOLATED FROM BEEF CORNEA B E R N A R D WORTMAN Department of Ophthalmology, Washington University School of Medicine, St. Louis, Mo. ( ~..... I.) (Received April Ist, I964)

SUMMARY

Glycosaminoglycans were isolated from beef cornea and further fractionated on the basis of ethanol solubility. Variability of keratan sulfate was found. The forms differ in reducing activity, sulfur content, infrared spectra and specific optical rotation. Keratan sulfotransferase (3'-phosphoadenylylsulfate:keratan sulfotransferase) activity is low but detectable in beef cornea-epithelial extract. Keratan sulfate with the lower sulfate content acts as a sulfate acceptor while the other does not.

INTRODUCTION

Glycosaminoglycan sulfotransferases (3'-phosphoadenylylsulfate glycosaminoglycan sulfotransferases) catalyze the transfer of sulfate from PAPS to acceptor polymers with some degree of substrate specificity. Although the existence of chondroitin and heparin sulfotransferases have been verified, keratan sulfotransferase activity has not been detected 1-4. Keratan sulfate has failed to show significant sulfate acceptor properties when used with various tissue extracts which are known to contain sulfotransferases ~. The past failures to demonstrate keratan sulfotransferase activity may be due to the use of tissues which do not normally contain keratan sulfate and thus lack the capacity for its synthesis. Cornea contains keratan sulfate, chondroitin 4-sulfate and chondroitin s, and so, it should also contain keratan and chondroitin 4-sulfotransferases. It is known that keratan sulfate from various connective-tissue sources has shown differences in peptides, sialic acid and methyl pentose bound to the glucosamim,glycan. Keratan sulfate and chondroitin 6-sulfate appear to be complexed with the same protein in cartilage. There are indications that keratan sulfate and chondroitin 4-sulfate do not interact in the same manner in cornea~L The present communication deals with observations concerning keratan sulfate isolated from beef cornea. Two forms of keratan sulfate which differ in sulfate-acceptor properties can be isolated from cornea. The sulfate-acceptor properties appear to be related to some physical and chemical characteristics of keratan sulfate and to the presence of keratan sulfotransferase activity in beef-cornea-epithelial extract. -Abbreviations: PAPS, 3'-phosphoadenosine 5'-phosphosulfate; PAl', 3'-phosphoadenosine 5'-phosphate. Biochim. Bioph.vs..4cta, 83 (1964) z ~ - ' 9 5

KERATAN SULFATE VARIABILITY

Z89

EXPERIMENTAL PROCEDURE

Chemicals Ecteola, p-nitrophenyl sulfate and p-nitrophenol were obtained from Sigma Chemical Company, St. Louis, Mo. PAP was isolated from Pabst ADP lot No. 6o2 and an additional supply was obtained from Dr. D. LIPKIN, Department of Chemistry, Washington University*,e.

Preparation of glycosaminoglycans Corneal glycosaminoglycans were isolated by ethanol precipitation after proteolytic digestion of beef cornea, as previously described~, 5,7. These glycosaminoglycans were further resolved into 5 fractions on the basis of ethanol solubility (Fig. x).

Anion-exchange chromatography Ecteola with a capacity of 0.3 mequiv/g was washed in x N NaOH, water, and dried with ethanol and ether e. Columns were packed under 6 lb/in z pressure and washed overnight with o.05 N HCI. Glycosaminoglycans were applied and eluted with a linear salt gradient 7.

Infrared spectra, sedimentation coetFzcientand optical rotation Keratan sulfates were incorporated into KBr discs and infrared spectra obtained with the Perkin-Elmer spectrophotometer model zx. Sedimentation coefficients were measured on the Spinco Model-E ultracentrifuge. Optical rotation was measured on a high precision polarimeter (O.C. Rudolph and Sons, Caldwell, N.J.).

Preparation of epithelial extract Epithelium was scraped from fresh beef corneas, homogenized in 4 vol. (w/v) of 5 mM Tris-HCl (pH 7.6) and centrifuged at 348oo × g for x h (see refs. 4, 6). The supernatant fluid was used immediately or stored at --zo °. All operations were conducted in the cold (o--4°), unless otherwise stated.

Enzyme assay Sulfotransferase activity measurements are based on the enzymatic formation of p-nitrophenol from p-nitrophenyl sulfate. Epithelial extracts also contain phenol sulfotransferase (EC 2.8.2.I) 4. The reaction scheme is as follows: PAP + p-nitrophenyl sulfate ~--pPAPS + p-nitrophenol PAPS -i- glycosaminoglyca~ ~ PAP + sulfated glycosaminoglycsn

(x) (2)

Reaction r serves as the "feeder-system" for Reaction 2. RESULTS

Step-wise ethanolfractionation The fractions obtained by ethanol fractionation were analyzed for hexose and hexuronic acid content T (Fig. x). It was further verified by anion-exchange chromatography that these fractions contained: chondroitin and chondroitin sulfate (I and II); chondroitin, chondroitin sulfate and a trace amount of keratan sulfate (III). In addition, the major portion of keratan sulfate was divided into two fractions, IV and V (Table I). Biochira. Biophys. Acta, 83 (x964) 288-295

290

B. WORTMAN Beef cornea glycosaminoglycans: ( 0 Dissolve in 5 % sodium a c e t a t e - o . 5 N acetic acid (2) Bring to 4 0 % ethanol (3) S t a n d 24 h a t 4 ° (4) Centrifuge a t 348oo x g for zo rain

t Supernatant: (x) Bring to 5 0 % ethanol (2) S t a n d 24 h at 4 ° (3) Centrifuge at 34 8oo × g for 2o min

Insoluble (I)

I

i

--I I

Insoluble: (I) Dissolve in 5 % calcium a c e t a t e - o . 5 N acetic acid (2) Bring to 4 ° % ethanol (3) S t a n d 24 h a t 4 ° (4) Centrifuge at 348o0 × g for 20 rain

Soluble: recovered a t reduced pressure (1) Dissolve in w a t e r (z) Bring to 6 o % ethanol (3) Centrifuge a t 348oo × g for : o rain t

. . . . . J

Insoluble (111)

Soluble: recovered at reduced pressure (IV)

Supernatant: Insoluble (II) (I) Bring to 6 o % e t h a n o l (2) S t a n d z4 h at 4 ° (3) Centrifuge a t 34 8oo × g for 2o rain _ Insoluble (V)

.

T T

S u p e r n a t a n t : discarded

Fig. L Procedure for e t h a n o l fractionation of beef corneal glycosaminoglycans. TABLE I TYPE

OF GLYCOSAMINOGLYCAN

FOUND

AFTER

ETHANOL

FRACTIONATION

OF BEEF CORNEAL GLVCOSAMINOGLYCANS T h e fractions were o b t a i n e d b y the scheme shown in Fig. I. Glycosaminoglycan c o n t e n t of fractions verified b y resolution on Ecteola columns a n d analysis for hexose a n d hexuronic acidL Glycosaminoglycan Fraaiwa

C& m d ~ in

Chomgroilin ~4foU

I

+

+

1I 111 IV V

+ ÷

-6 +

Keralan s,dfaU

trace -i+

Anion-exchangechromatography The two keratan sulfate fractions and a fraction free of keratan sulfate (IV, V and II, Table I) were applied to and eluted from Ecteola columns e. Keratan sulfates, Fractions IV and V, were eluted with o . i - o . 5 and with o.3--o.8 M NaCI, respectively Biochim. Biophys. Acta, 83 (t964) 288-z95

KERATAN SULFATE VARIABILITY

29][

(Fig. zA). Fraction II contains hexuronic acid and appears to be a mixture of chondroitin and chondroitin sulfate (Fig. 2B). t

~40

I.O

4t z, i

iR

° do

zoo 3oo £ f ' f ~ Vo~ms,mt

s

o

Fig. 2. Resolution of glycosaminoglycans on Ecteola columns. Giyco~mlnoglycans were &pplied to the columns (x x a 5 cm) a n d eluted with o.o5 N HCI, followed b y a linear gradient to z M NaCI in o.o 5 lq HCI. 5-ml volumes of effluent were collected a t the rate of 3o mlIh a n d analyzed for hexose a n d hexuronic acid content. A, composite diagram of elution 1~ttern ob~t=-ined h-ore two columns. Fraction IV ( O - - O ) , xo.8 mg applied; Fraction V ( @ - - @ ) , 7.4 mg applied. B, Fraction II (=----ms), 9-4 mg applied.

Infrared spe,ctra Infrared spectra of authentic keratan sulfate (supplied by T. C. LAUee~rr, Uppsala University, Sweden) and keratan sulfate IV showed identical absorption bands (Figs. 3A and B). Keratan sulfate V demonstrated an additional absorption band at 7.2/t, and other quantitative differences are seen at 8.z, 6.4 and 3.5/~ (Fig. 3C). These absorption bands may be attributable to sulfate and acetylamino groups.

Composition of kera~an sulfate The two keratan sulfate fractions (IV and V, Table II) were analyzed for chemical constituents and found to differ in their reducing activitiess, chain weights', and sulfur content (sulfur analyses were performed by Micro-Tech Laboratories, Skokie, Ill.). Keratan sulfates IV and V contain 4.5 and z.5 % sulfur, and o.z and T A B L E II COMPOSITION

OF KRItATAN

SULFATB

FRACTIONS

The fractions correspond with those shown in Fig. z. /v % hexoae (see ref. 7) % h e x u m n i c acid (see tel. 7) % hexosmmine (see ref. 2z) % nitrogen (micro-Kjeldahl) % sulfur (Micm-Tech Lab.) % reducing sugar (see ref. S) sit0, w

30.0 3 .2 24.4 3.2 4.5 o.2 l.II

v 3t.2 4.5 24.6 3.5 2.5 0.4 1.2 5

~

+ xo.5°

+0.8 °

Chain weight (see ref. 9)

52.8" Io a

25.5" xos

Biockim. Biophys. dcta, 83 (x964) 288-295

:292

B. WOI~TM.~N

~' t-i

•

'

"

/i

"

~

::;! i

.

.

.

.

4--4,.

! ~,~:

, ~:i ~

,,

I",

,

~--

'

• -¢--~"

I

!

i i

:

•

i

!

r

'

!

I

,

'

!-i i ri

-~

;:! i ~i

• i--~--

~ - ~ - - - *

i

I-il ~

! i---i

'

!

~-

+

, :Ii

4---+'--~

"

i--,-i

--'r"

-~

~

-'I

! j,_L.~.~' ~ ' ~--r--~-~' ~'---4-

)

w~vt~

.

::i

i:-

~

r

'

~ :

i'I

"

I"

,.~

.:

m

i 1 \l L,,.!_L_.L.~.. ;A._!__L ~-A-~F-~.- L-I J~,4~-I

l.

I ~

: ....

~

. . . . . . . . . .

- L/~. ] ~ :~ '

....

k~l ~ : = -

')

:=

[o

~ ~'~: ~ ~:

Fig. 3. Infrared spectra of keratan sulfates. A, authentic keratan sulfatO C, keratan sulfate V.

: , - . ~~ - - ~

~

u

B, kt'r;ttal| sulf;tte IV.

A

B Fig. 4. Schlieren p h o t o g r a p h s of k e r a t a n sulfate. T h e direction of s e d i m e n t a t i o n i~ from r i g h t to left. R u n s were m a d e a t 59780 r e v . / m i n in o. t5 M KCI. B a r a n g l e s were 7o''. A, k e r a t a n s u l f a t e I V: p i c t u r e s were t a k e n a t z i , 36, 54, 69, 8i a n d 90 rain a f t e r r e a c h i n g full speed. B, k e r a t a n s u l f a t e V : p i c t u r e s were t a k e n a t 21, 39, 63, 8t, xoz a n d t t 4 rain a f t e r reztcbiqg full speed.

Biochim. Bioph.vs. Iota. 83 (1~J4) z~,~ 295

KERATAN SULFATE VARIABILITY

293

0.4% reducing sugar, respectively. Sedimentation coefficients of x.xx S and x.z5 S, and specific optical rotation values of Jr xo.5 ° and +0.8 ° were obtained for keratan sulfates IV and V, respectively (see Fig. 4).

Glycosaminoglycan and PAP dependency The production of p-nitrophenol from p-nitrophenyl sulfate is shown to be dependent upon PAP in the presence of phenol sulfotransferase which is present in the extracts'. There is an appearance of p-nitropbenol in the presence of the unresolved mixture of cornea] glycosaminoglycans and Fractions II and V, but not in the presence of Fraction IV (Fig. 5). These enzymatic activities are indicative of keratan and chondroitin 4-sulfotransferases. 1.4

'0

to

I

I

I

I

I

I

I

i

t

I

I

I

-D,

•

t(

J

O.E 02 t 3'" Pholpl~oodeno,.ine

5'- PhOslPhale.mpmo~es

Fig. 5. p-!qitrophenol production dependent upon PAP and glycosaminoglycans. Incubations contained: x9.5/~moles of phosphate buffer (pH 7.o); 45.8 m/Amoles of p-nitrophenyl sulfate; 243 pg of epithelial extract protein; varying amounts of PAP in a total volume of x24pl and incuhated for 2 h at 38°. A, unfractionated corneal glycosaminolglycams, 434/Ag. B° Fraction II, 39x pg. C, ker~tan sulfate V, 399/~g. D, keratan sulfate IV, 297/~g. Q m Q , complete system; O - - O , glyco~minoglycan omitted.

Effect of time, protein and keratan sulfate concentrations In the presence of PAP and keratan sulfate V, the production of p-nitropbenol reached an equiSbrium by the second hour at 38 ° (Fig. 6A) ; the reaction is linear up to the addition of approx. 3oo/~g of protein (Fig. 6]3) and maximal in presence of approx. 3/tg of keratan sulfate V per/~1 of reaction mixture (Fig. 0C). In the absence of added PAP, and in the presence of keratan sulfate, the production of p-nitrophenol is detected after 2 h at 38 °. DISCUSSION

This is the first report of keratan sulfotransferase activity and of a variability in keratan sulfate isolated from beef cornea. The keratan sulfate previously obtained Biochim. Biophys. Acta, 83 (x964) 288-295

294

B. WORTMA.~

after resolution of corneal glycosaminoglycans by anion-exchange chromatography did not demonstrate a low sulfate content n ; a sample of that keratan .sulfate (supplied by T. C. LAURENT, Uppsala University, Sweden) was used as the standard for infrared spectroscopy. 'B.

°'fA HOu~ el ~1"

o

~o

' 3bo ' .~bo

I~'elein.pll

o 2 4 6 B t¢4~1o,~uel*,p41~

Fig. 6. Effect of time, protein a n d keratan sulfate V concentration on p-nitrophenol production. At the end of all incubations, 6. 5 pl of 5 N NaOH were added a n d absorbancy read a t 400 m p a n d compared with p-nitrophenol standards. A, incubations contained: I9.5//moles of phosphate buffer (pH 7.0); 45.8 mpmoles of p-nitrophenyl sulfate; 276pg of epithelial extract protein: 367 p g of keratan sulfate V; 0.32 mpmole of P A P in a total volume of 122 pl. B, incubations contained: 19.5/,moles of phosphate buffer (pH 7.o); 45.8 mpmoles of p-nitrophenyl sulfate: 507 pg of keratan sulfate V; 0.32 mpmole of PAP; varying amounts of epithelial extract protein in a total volume of I58 pl. Incubations were for 2 h a t 38°. C, incubations contained I9. 5/~moles of phosphate buffer (pH 7.0) ; 45.8 mpmoles of p-nitrophenyl sulfate: 243 pg of epithelial extract protein; varying amounts of keratan sulfate V; 0.58 mpmole of P A P in a total volume of i37 Itl. Incubations were for z h at 38°. A - - A , complete system; O - O , keratan sulfate omitted: • - - • , P A P omitted.

Keratan stdfate is a polydispersed material, and its molecular weight will vary with the tissue source n. Keratan sulfate from nucleus pulposus has an estimated molecular weight of I5 ooo-zoooo while that from cornea had an estimated molecular weight of 8700--19100. Chondroitin 4-sulfate is a higher-molecular-weight species of glycosaminoglycan; its molecular weight ranges from 39000 to 43000. The polydispersity of molecular weight and/or charge density of glycosaminoglycans has been observed previouslyll, TM. Cornea contains keratan sulfate, chondroitin 4-sulfate and chondroitin 6. After the discovery of chondroitin in cornea, it was postulated that this is the low or unsulfated precursor of chondroitin sulfate is. According to this concept, the polymer is synthesized prior to sulfation or the synthesis is a multistep process in which sugar units as well as sulfate groups are added to polymer-chains of small size.This implies that for each sulfated glycosaminoglycan found in a particular tissue there should also exist a precursor polymer. Subsequent investigations have supported this hypothesist4, is. However, the possibility of finding the precursor polymers in detectable quantities may in part depend upon equilibrium kinetics. Chondroitin 6-sulfate exists in connective tissues as a protein-polysaccharide complex which usually contains trace amounts of keratan sulfate is. It remains a speculative hypothesis to be proven whether keratan sulfate and chondroitin 4-sulfate are complexed with the same protein or exist as separate entities in cornea ~7.~2. Corneal glycosaminoglycans are in an active metabolic state and are turning-over at a very slow but known rate is. Es'~SlSulfate ions are incorporated into chondroitin and keratan sulfates in vivo and in vitro and are the basis of half-life measurements tg. Keratan sulfate in cornea Is and in nucleus pulposus ~° has a long half-life which may Biochim. Biophys..4eta, 8 3 (t964) 288-295

KERATAN SULFATE VARIABILITY

295

be greater than xzo days in the latter structure. Multiple labeling experiments have suggested that the polysaccharide, sulfate and complexed-protein moieties axe synthesized as a unit. The incorporation of sulfate into the polymer probably represents actual lengthening of the polysacchaxide chain, i.e., a true synthesis and not an exchange reaction. It is probable that sulfate is not transferred to a fully formed glycosaminoglycan but to a relatively small molecule which is further polymerizedS,l*, is. It is possible that the above mentioned mechanism, previously proposed for the biosynthesis of chondroitin sulfate by embryonic chick cartilage ~* and that for the sulfation of heparin Is, may hold true in part for the biosynthesis of keratan sulfate by cornea. ACKNOWLEDGEMENTS

The author thanks Mrs. F. BLACKfor technical assistance, and Mr. A. LEURE-DuPREE for ultracentrifugal analyses. This investigation was supported in part by U.S. Public Health Service Research Grant HB-ox 9xi from the National Institute of l~leurological Diseases and Blindness and by the St. Louis Heart Association. This work was done during the tenure of an Established Investigatorship of the American Heart Association. REFERENCES

E. D. KORN, J. Biol. ¢ ~ m . , 254 {I959) z647. s S. SuzuKI AND J. L. STnOMINOEa, J. Biol. Chem., 235 096o) 257. s E. A. DAVIDSON AND J. G. RILRV, J. Biol. Chem., 235 (I96o) 3367. d B. WOR'rMAN,f . Biol. Chem., 236 (x961) 974. s K. MEYER, A. LINKER, E. A. DAVlDSON AND B. WIglSSMANN,J. Biol. Chem., 205 (I953) 6Xl. s B. WORTI~AN, Bioclsim. Biophys. Acta, 77 (I963) 65. B. WORTMAN, ANal. Biochem., 4 (I962) Io. s j . PARK AND W. JOHNSON.J. Biol. Chem., 18I (1949) I49. * J. C. HOUCK AND R. H. I~AnCZ, Biochim. Biopl~ys. Acla, 25 (I957) 6o 7. 10 S. SUZUKI AND J. L. STROMINOER, J. Biol. Chem., z35 (I96o) 267, 11 T. C. LAURENT AND A. ANSETN, Exptl. Eye Res., x 096I) 99. za A. At~SBTH AND T. C. LAURENT, Exptl. Eye Res., x (I96I) 25. la E. A. DAVlDSON AND K. MEYI~R, J. Biol. Chem., 2II (1954) 605. 1* j . B. ADAMS, Biochem. J., 76 (196o) 520. Is L. SPOLTER, L. I. RIcE ASD W. MARX, Biochim. Biophys. AttN, 74 (x963) I88. " J. D. GBEOORY AND L. RODtN, Biochem. Biophys. Res. Commun., 5 (1961) 43o. xT E. A. DAVIDSONAND W. SMAI.L, Bio~him. Biopkys..4cla, 69 (1963) 459. 18 C.-H. DOHLMAN AND H. ]~OSTROM,Acta Ophthalmol., 33 (1955) 455. t, B. WonTUAN AND J. L. STROMINO~R, A ~ . J. Ophthalmol., 44 (I957) 291. E. A. DAVlDSON AND W. SMALL, Biochim. Biophys. ,4cla, 69 (I96]) 445. at C. J. M. RONDLE AND W. T. J. MOltGAS, Biochem. J . , Ol (I955) 586. ~t K. MsYeR, N. SENO, B. ANDERSON, V. LIPPMAN AND 13. HOFFMAN, Fedevatio~ Proc., 23 (i964) abstract 2295. Biochim. Biophys. Acta, 83 (1964) 288-295