Structures of N- and O-linked oligosaccharides from chondrosarcoma proteoglycan

OSTEOARTHRITIS SYMPOSIUM

12

rabbit serum was used instead of the immune rum (lanes 2 and 4).

antise-

CONCLUSIONS

The core protein of cartilage proteoglycan as an intermediate in the biosynthesis of proteoglycan by intact chondrocytes has been tentatively identified. It appears to be largely a monodisperse species of M, -

370,000 on SDS-PAGE. The presence of a single predominant core protein intermediate suggests that proteoglycan synthesis proceeds through a rate-limiting step that allows the accumulation of label in this intermediate. The nature of this step is not known, but it may represent the transport time for the movement of newly synthesized core protein from the rough endoplasmic reticulum to the Golgi for the addition of chondroitin-sulfate chains.

REFERENCES I. Kimura JH, Caputo CB, Hascall VC. cycloheximide chondrocytes

The effect of

on synthesis of proteoglycans from the Swarm

by cultured

rat chondrosarcoma.

J Biol

Chem (in press).

clostripain

digestion of proteoglycans

chondrosarcoma. 3. Kimura Poole AR.

2. Caputo CB, Hascall

VC.

Maccallum

DK,

Characterization

Kimura

JH, Schrode J,

of fragments

produced

Structures of N- and O-linked chondrosarcoma proteoglycan

by

JH, Thonar

Identification

proteoglycan

rat

Reiner

A.

of core protein, an intermediate

EJ-MA,

in

biosynthesis,

from the Swarm

from the Swarm

Arch Biochem Biophys 1980;204:220-33. Hascall

made by cultured

rat chondrosarcoma

VC,

chondrocytes

(unpublished

manu-

script).

oligosaccharides

from

By S. Lohmander, B. Nilsson, S. De Luca. and V. Hascall; the Laboratory of Biochemistry, National Institute of Dental Research, Bethesda, Maryland, and Department of Physiological Chemistry, University of Lund, Sweden

T

HE PROTEOGLYCANS of the transplantable Swarm rat chondrosarcoma have been extensively characterized’-* and have been shown to differ from the proteoglycans of hyaline cartilage in that they lack keratan sulfate. Recent studies have however shown that the chondrosarcoma proteoglycan in addition to chondroitin sulfate chains contains two additional classes of carbohydrate substituents, namely N- and O-linked oligosaccharides. This report presents additional information on the structure of these oligosaccharides. MATERIALS AND Proteoglycan coma

subjected

by trifluoroacetolysis4

and

to specific chemical degradations. RESULTS

In a previous investigation3 we isolated a family of O-linked oligosaccharides from the rat chondrosarcoma proteoglycan. In the present study we were able to confirm the proposed structure for the largest of these oligosaccharides by direct probe mass spectrometry of the desialylated, permethylated derivative which gave a fragmentation pattern consistent with the structure shown in Fig. I. The N-linked oligosaccharides were resolved into

monomer was isolated from rat chondrosar-

and the oligosaccharides

chromatography and linkages

METHODS

were released from the protein

as described.’

were determined

analysis

before and after

O-linked

oligosaccharides,

from the protein,

purified

by repeated

Monosaccharide

by sugar and methylation

removal

of sialic acid.

released by alkaline The

N-linked

For the

borohydride

the sequences were determined

probe mass spectrometry.

gel

composition

by direct

oligosaccharides

Supported in part (S.L.) by the Swedish Medical Research Council Grant K80-I7P-5820 and B8/-13X05948. 0 1981 by Grune & Straiton. Inc. 0049-0172/81/1005~005$01.00/0

NIINP12 y~3Gal, p_~GlcN~~, IIfZMan, 0 Fig. 1. Structures of 0- and N-linked oligosaccharide isolated from chondrosarcoma proteoglycan.

OSTEOARTHRITIS

SYMPOSIUM

13

two peaks by chromatography on BioGel P-10. Analyses for neutral sugars, hexosamines and sialic acid of the larger, predominant N-linked oligosaccharide gave ratios of sialic acid:fucose:mannose:galactose:N-acetylglucosame of 3:1:3:3:5. Methylation analysis of this oligosaccharide and subsequent hydrolysis, reduction, acetylation and gas-liquid chromatography/mass spectrometry gave the results shown in Table I. The observed partially methylated alditolacetates are consistent with the structure shown in Fig. I. This tri-antennary oligosaccharide accounted for about 80% of the N-linked oligosaccharides. Analyses of the second, smaller peak indicated the presence of a biantennary analog. DISCUSSION The proteoglycan monomer from the Swarm rat chondrosarcoma contains two different mannoseTable 1. Methylation Analysis of N-Linked OQgosaccharide* TFAtlTFAAS Untreated

llloa

0.59

0.67 2.61 -

2.3,4 Fuc 2.3,4.6 Gal 2,4,6 Gal 3,4,6 Man 3,6 Man 2,4 Man 3,6 GlcN( Me)Ac 3 GlcN (Me)Ac 1,3,5 GlcN (Me)Ac *Values tTFA

3.07 0.97 1.04 1.oo + + -

0.66 1.03 1.00 + +

given as molar ratios.

= Trifluoroacetic

acid.

$TFAA = Trifluoroacetic

anhydride.

containing, N-linked oligosaccharides and a family of at least three different O-linked oligosaccharides. An average proteoglycan molecule would contain a total of about 10-l 5 N-linked oligosaccharides, 110 O-linked oligosaccharides and 85 chondroitin sulfate chains. The N-linked oligosaccharides are located in or near the globular hyaluronic acid binding region of the protein backbone while the O-linked oligosaccharides are distributed outside this region. The N-linked oligosaccharides found in the proteoglycan are of the complex type and are analogous in structure to those found, for example, in serum glycoproteins. The O-linked oligosaccharides are on the other hand structurally similar to those usually found in glycoproteins from epithelial secretions, mucins, but also in other glycoproteins like fetuin and epiglycanin. The heterogeneity in structure of the O-linked oligosaccharides in the proteoglycan probably represents varying degrees of completion of the same primer. The linkage region for attaching cartilage keratan sulfate chains to core protein is similar in structure to the O-linked oligosaccharides which therefore may represent initiated but unfinished keratan sulfate chains. Indeed, recent data indicate an inverse relationship between the number of O-linked oligosaccharides and keratan sulfate chains in proteoglycans from different cartilages and at different ages.3.5 The functional significance of oligosaccharides in proteoglycans as well as in glycoproteins is obscure. The presence of a considerable amount of carbohydrate substituent in the globular region of the core protein is however of interest in view of the important role this part of the protein plays in proteoglycan aggregate formation by interaction with link protein as well as hyaluronic acid.

REFERENCES 1.

Oegema

Hascall VC, Dziewiatkowski

TR,

tion and characterization rat chondrosarcoma.

of proteoglycans

J Biol Chem 1975;250:615

2. Faltz LL, Reddi AH, Hascall from

VC. the

rat

with

extracted associative

solvents. J Biol Chem 1979;254: 1375-80. 3. Lohmander

LS, De Luca S, Nilsson

charides on proteoglycans from the Swarm rat chondrosarcoma. J

Biol Chem

1980;255:6054-9

tion of the protein carbohydrate

part of glycoproteins-Isolation

chains

al. Oligosac-

of

asialofetuin.

of the

Carbohydr

Res

1979;72: 183-9. 5. Sweet MBE, Thonar EJ-MA changes in proteoglycan

B, et

I.

4. Nilsson B and Svensson S. New method for degrada-

D, Pita JC,

of proteoglycans

chondrosarcoma

Isola-

l-9.

Hascall GK, Martin

Characterization

Swarm

DD.

from the Swarm

1979;198:439%48.

structure.

and Marsh J. Age-related Arch

B&hem

Biophys