Chondroitin 4-Sulfate Covalently Cross-links the Chains of the Human ...

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Jul 26, 1990 - Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010. The human blood protein pre-a-inhibitor is composed.

THEJOURNALOF ( C

Vol . 266, N O . 2, Issue of January 15, pp. 747-i51, 1991 Printed in (1.S. A .

BIOLOGICAL CHEMISTRY

1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Chondroitin 4-Sulfate Covalently Cross-links the Chains of the Human Blood Protein Pre-winhibitor” (Received for publication, July 26, 1990)

Jan J. Enghild$§,Guy Salvesen$, Stanley A. Heftall, IdaB. Thegemen$, Shane Rutherfurdv, and Salvatore V. Pizzos From the $Department of Pathology, Duke Uniuersity Medical Center, Durham, North Carolina 27710 and the TDiuision of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010

The human blood protein pre-a-inhibitor is composed of one heavy and one light protein chain. The chains are covalently linked to each other by a structure that has not previously been described, which we designate a protein-glycosaminoglycan-protein (PGP) crosslink. A combination of protein and carbohydrate analytical techniques indicates that the interchain linkage is mediated bya chondroitin 4-sulfate glycosaminoglycan that originates from a typical 0-glycosidic link to Ser-10 of the right chain. The heavy chain is esterified, via the a-carbon of its C-terminal Asp, to C-6 of an internal N-acetylgalactosamine of the glycosaminoglycan chain. This PGP cross-link may be present in other proteins, but could have been overlooked due to the heterogeneous behavior of proteins containing glycosaminoglycan.

The most common interchaincross-linkinproteinssecreted from cells is the disulfide formed following oxidation of cysteine residues. In mammals, a number of less common cross-links, usually resulting in the precipitation of soluble proteins, have been identified. These include the glutamyllysyl cross-link of transglutaminases (1);the desmosine and isodesmosine cross-links, formed following condensation of oxidized lysine residues, that stabilize mature elastin (2);and the pyridinoline and lysinonorleucine cross-links that join lysyl or hydroxylysyl side chains of collagen monomers (3). It is generally assumed that these cross-links impart structural rigidity to fibrin and connective tissue proteins. It is unusual to find non-disulfide covalent cross-links in soluble blood proteins. Nevertheless, we have recently shown (4) that the humanblood protein pre-a-inhibitor (PaI)’ consists of protein chains that contain a carbohydrate cross-link. Pa1 is composed of a heavy chain of -80 kDa and a light chain of -30 kDa (4). The heavy chain is called HC3 to distinguish itfrom the two heavy chains of the related protein * This work was supported in partby National Instituks of Health Grants CA-33572 and HL-24066. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Partially supported by a grant from the Danish Research Academy. To whom correspondence should be addressed. ’ T h e abbreviations used are:POI,pre-a-inhibitor; IaI, inter-ainhibitor; EDC, l-ethyl-3-(dimethylaminopropyl)carbodiimide; FABMS, fast atom bombardment mass spectrometry; GAG, glycosaminoglycan; PGP,protein-glycosaminoglycan-protein;PVDF, polyvinylidene difluoride; PTH, phenylthiohydantoin; RP-HPLC, reversephase high performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

inter-a-inhibitor ( I d ) (4-6), andthelightchainis called bikunin since it consists of two pancreatic trypsin inhibitor (Kunitz)-type domains (6). Little is known of the biological importance of Pa1 since it has only recently been described as a distinct protein (4). Possibly, the function is related to that of I d , although the role of the latter is not clear ( 7 ) , despite its ability to inhibit a number of serine proteinases. Pa1 migrates as a single band on SDS-PAGE, even in the presence of reducing agents, but is separated into its constituent protein chains following treatment with hyaluronidase or chondroitin ABC lyase (4). This suggests that Pa1 is assembled by a chondroitin-like glycosaminoglycan (GAG) cross-link. To elucidate the structure of the cross-link, we have subjected Pa1 to limited proteolysis to obtain peptide fragments that have been analyzed by peptide sequencing, amino acid andcarbohydrate compositionalanalyses, fast atombombardmentmassspectrometry(FAB-MS),and monoclonal antibody analysis of derivatives. EXPERIMENTALPROCEDURES

Materials-Chondroitin ABC lyase, 1,lO-phenanthroline, tetrazotized o-dianisidine (fast blue salt B), N-acetyl-DL-phenylalanine pnaphthylester, 5-bromo-4-chloro-3-indolyl phosphate,nitro blue tetrazolium, glycinamide, and l-ethyl-3-(dimethylaminopropy1)carbodiimide (EDC) were from Sigma. Bovine trypsin, Staphylococcus aureus V8 proteinase, 3,4-dichloroisocoumarin, and N-[N-(L3-trans-carboxyoxiran-2-carbonyl)-~-leucyl]-4-aminobutylguanidine were from Boehringer Mannheim. Rabbit anti-mouse alkaline phosphatase-conjugated antiserum was from Bio-Rad. Monoclonal antibodies against unsulfated, 4-sulfated, and 6-sulfated chondroitin were from Saikagaku. Pa1was purified as described before (4). SDS-PAGE-This was performed on 5-15% gradients gels using the glycine/2-amino-2-methyl-1,3-propanediol/HC1 system described previously (8). The gels were stained for protein using Coomassie Blue or for trypsin inhibitor activity employing a counterstain procedure (4,9). Immunoblotting-Pa1 was digested with chondroitin ABC lyase (4), separated by SDS-PAGE, and electroblotted to polyvinylidene +fluoride (PVDF)membranes (10). The reactivity of monoclonal antibodies which specifically recognize unsulfated, 4-sulfated, and 6sulfated chondroitinwas investigated. The bands containingepitopes recognized by the antibodieswere visualized using a secondary rabbit anti-mouse alkaline phosphatase-conjugated antiserum (4). Purification of Cross-link-containing Peptides-Pa1 was digested with thermolysin using an enzyme/substrate ratio of 1:250 (mole/ mole) in 50 m M Tris-C1, 0.1 M NaCl, 2 mM CaC12,pH 7.4, for 30 min a t 37 “C. The reaction was stoppedby adding 1,lO-phenanthrolineto a finalconcentration of 1 mM. The fragments were separated by reverse-phase high performance liquid chromatography (RP-HPLC) using a C, reverse-phase column (Aquapore RP-300, Brownlee Labs) and the HPLC systemdescribed previously (4, 11). The column was equilibrated with 5% acetonitrile and 0.1% trifluoroacetic acid and developed with a gradient to 90% acetonitrile a t 2% min“ and a flow rate of 1 ml min”. The 45-kDa polypeptide containing trypsin inhibitory activitywas collected,concentrated by freeze-drying, and further digested for 40 min at 37 “C using S. aureus V8 proteinase at an

747

Novel Glycosaminoglycan-mediated Protein Cross-link

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enzyme/substrate ratio of 1:40 (w/w) in NH,HC03. The reaction was terminated by the addition of 3,4-dichloroisocoumarin to a final . fragments were purified by RP-HPLC concentration of 50 p ~ The as described above. Amino Acid Analysis-Peptides (1-2 nmol) were hydrolyzedin 6N HCI for 24 h a t 110 “C (12). The hydrolyzed samples were dried in a SpeedVac concentrator (Savant Instruments, Inc.), and the amino acid composition was determined in a Beckman Model 6300 amino acid analyzer using the sodium citrate buffers provided by the manufacturer. Carbohydrate Compositional Analysis-Samples were hydrolyzed in 2 M aqueous trifluoroacetic acid or 6 N HCI. The tubes were sparged ofoxygen with helium, sealed, and hydrolyzed for 4 h a t 100 “C. Following hydrolysis, the samples were analyzed with a Dionex BioLC system equipped with an amperometric detector. Protein Sequence Analysis-Automated Edman degradation was carried out in an Applied Biosystems Model 477A Sequencer with on-line phenylthiohydantoin (PTH) analysis using an Applied Biosystems Model 120A HPLC apparatus. The instruments were operated as recommended in the user bulletins and manuals distributed by the manufacturer. Some samples were separated by SDS-PAGE and electroblotted to PVDF membranes prior to sequence analysis (10). FAB-MS-Samples were dissolved in 2 pl of distilled deionized water and added to 2 pl of 10% ethanolamine in glycerol (v/v) onthe stainless steel sample stage. Spectra were obtained using a JEOL HX-100HF double-focusing magnetic sector mass spectrometer operating a t 5-kV accelerating potential with a nominal resolution of 500. Sample ionization was accomplished using a 6-keV Xenon atom beam. The electric sector was set to transmit all ions a t source potential (5 kV), and the magnetic sector was scanned over a given mass range. Deglycosylatwn with NaOH-Intact Pa1 (50 pg) or the C-terminal peptide (1-5 nmol) (see Table I, column 4a) was desalted by dialysis into water or by RP-HPLC as described above, followed by freezedrying. The samples were dissolved in 100 pl of 50 mM NaOH at 23 “C and applied to SDS-PAGE or RP-HPLC after 5 min. This procedure dissociated Pa1 to give HC3 and bikunin withoutany detectable degradation of the protein. The C-terminal Asp was positively identified during sequence analysis, indicating that the NaOH treatment removed the carbohydrate from that residue. Other Methods-Treatment of the C-terminal peptide with EDC and glycinamide was performed as described before (13). RESULTS

Isolation of Cross-linked Peptides-Limited digestion of Pa1 with thermolysin resulted in removal of the majority of the heavy chain to generate bikunin-containing fragments of 45 and 22 kDa(Fig. 1). We note that the 45- and 22-kDa fragments visible in Fig. l b do not stain well with Coomassie Blue (Fig. la). Presumably, GAG interferes with staining of the 45-kDa fragment. The amount of the 22-kDa fragment generated was much lowerthan that of the 45-kDa fragment and not enough to show up by CoomassieBlue staining. Protein sequence analysis of these two trypsin inhibitory fragments, after transfer from SDS-PAGE to PVDF mem-

a 190-

b

M 1 2 13 2 3

pgmmq

976643 31

21 14-

FIG. 1. Limited thermolysin digestion of PaI. Samples of Pa1 or thermolysin-digested Pa1 were run on SDS-PAGE, and the gels were stained with Coomassie Blue (a) or were stained for trypsin inhibitory activity (b). Lane I , PaI; lane 2, thermolysin digest; lane 3, RP-HPLC peak containing trypsin inhibitory activity.

brane (lo), revealed that the22-kDa fragment begins at Val19 of bikunin, whereas the 45-kDa fragment consists of two chains (see Table I, column 1).The sequences of the two chains correspond to a 17-residuepeptide of HC3 attached to the bulk of bikunin. The 45-kDa derivative, which contains the cross-link, was purified free of excess heavy chain derivatives by RP-HPLC. This material was further digested with S. aureus V8 proteinase to remove the majority of bikunin (downstream of Glu-18) (14). The digest was fractionated by RP-HPLC, giving two fractions, one of which contained two peptide sequences which terminated in cycle 18 (see Table I, column 2). This fraction was digested withchondroitin ABC lyase, and the digest was rechromatographed by RP-HPLC. Two fractions were recovered, and their combined sequences (see Table I, columns 3a and 4a) corresponded to the single fraction before chondroitin ABC lyase digestion (see Table I, column 2). This indicates that thepeptides contain the chondroitinase-sensitive cross-link of PaI. Position of Cross-link-The composition of each peptide was determined by amino acid analysis (see Table I, columns TABLE I N-terminal sequence analysis and composition of peptides containing the cross-link A portion of the 45-kDa fragment releasedfrom Pa1 following digestion with thermolysin was sequenced using an Applied Biosystems Model 477A Sequencer, and residues identified in the first 20 cycles are shown in column 1. The remainder of the 45-kDa fragment was concentrated by freeze-drying, digested with S. aurew V8 proteinase, and run on RP-HPLC under the conditions described under “Experimental Procedures.” A heterogeneous fraction eluting -20% acetonitrile contained two N-terminal sequences (column 2) terminating in cycle 18. This material was concentrated by freeze-drying and allowed to react with chondroitin ABC lyase in the presence of a mixture of proteinase inhibitors (26) a t 37 “C for 12h. The digest was run on RP-HPLC; twohomogeneous peptide peaks were recovered and submitted to N-terminal sequence analysis (columns 3a and 4a). Portions of the material were hydrolyzed, and their amino acid compositions were determined using a Beckman Model6300 analyzer (columns 3b and 4b). Each peptide sample was prepared and sequenced a t least five times. The values in parentheses indicate the yield of each PTH-derivative in picomolesfrom a representative analysis. Amino acid residues identified” Chondroitinase fractions Cycle no. 45-kDa fragment (1)

s ; ~ ~ Bikunin-de~ ~ ~ sC-terminal fraction (2)

rived peptide (4a) (3b) (3a)

peptide (4b)

AL(378,212) AL(8,14) A (339) A L(229) L VT (503,108) VT (16,8) V (473) V T (154) T LS (221,34) LS (14,8) L (203) L S (60) S PY (140,73) PY (14,9) P (238) P Y (150) Y Q (160) Q (16) Q (107) Z Q (118) Z E P (112,84) E P (6,141 E (113) Z P (155) P EP (120,63) EP(11,13) E (115) Z P (116) P EQ (116,42) EQ(11.8) E (190) Z Q (115) Z GN (96,35) GN (14,7) G (167) G N (71) B P (70) P (11) S P (86) P GY (97,41) GY (12,6) G (178) G Y (74) Y GY (112,55) GY (14,7) G (180) G Y (91) Y GY (103,51) GY (13,7) G (155) G Y (93) Y QV (54,371 QV (6,4) Q (40) Z V (66) V LD (71,23) LD (7,3) L (61) DL (41) B VG (71,41) VG (4,lO) V (50) V G (47) G T (31) T (3) T(16) T B E (21) E(10) E (2) Z V (50) T (32) ’Standard single-letter code is used. Compositionalanalysis following hydrolysis in HCI (columns 3b and 4b) does not distinguish between Asp and Asn (B) or Glu and Gln (Z). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Novel Glycosaminoglycan-mediated Protein Cross-link

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A 3b and4b). Residue 10 of the bikunin-derived peptide was the only residue not identified by peptide sequencing, but amino acid analysisindicated a Ser. This is consistent with the residue predicted from cDNA analysis and with this residue being glycosylated (14, 15). All sequence assignments of the HC3-derived peptide were confirmed by amino acid analysis, except that the compositional analysis demonstrated the presence of an Asx not present in the peptide sequence. The observed Asx represents an Asp, predicted by cDNA analysis (5),that would constitute the C-terminalresidue of the heavy chain-derived peptide. Since Ser-10 of bikunin and the Cterminal Asp of the heavy chain were only detected following hydrolysis in HCl, we conclude thattheseconstitutethe attachment sites for a covalent chondroitin ABC lyase-sensitive cross-link. Determination of Glycosaminoglycan Type-Carbohydrate analysis was performed on cross-linked material following digestion with S. aureus V8 proteinase (see Table I, column 2). Thecompositions demonstrated thepresence of glucuronic acidand GalNAc,with an average of 17 glucuronicacid saccharides/mol of peptide. A small amount of fucose was 0 0 0 detected, indicating someheterogeneity or a possible contam"0°C no-c "C inant. These compositions reflect a chondroitin-type struccn, 0 a, 0 7 8 ,*,,,"-L-! ture. The type of chondroitin was further investigated by ' 0 ' 0 ,""LE \O immunoblotting usingmonoclonal antibodies selective for I I various chondroitin species. Antibodies selective for unsulfatedchondroitinandchondroitin6-sulfate failed to react with the blot, but an antibody selective for chondroitin 4sulfate gave a clear reaction with the 30-kDa bikunin band 2124 (data not shown). The results of carbohydrate compositional and monoclonal antibody analyses are consistent with a chondroitin 4-sulfate structurefor the GAG cross-link. FAB-MS Analysis of Cross-link-Each peptide containing the attachment sites of the chondroitin 4-sulfate cross-link was analyzed by FAB-MS (see Table I, columns 3a and 4a). In this way, we were able t o ascertain the molecular compo2W. sition of the attachmentsites. The intact cross-linkfollowing FIG. 2. Negative ion FAB-MS of C-terminal peptide (see S. aureus V8 proteinase digestion (see TableI, column 2) was Table I, column 4a). A , spectrum in them/z 2000-2900 range, with treated with chondroitin AC lyase, and the resulting peptides the inset showing the interpretationof the structureof the molecular ion; B, fragmentation patterninvolving cleavages of the glycosyl ring. werepurified by RP-HPLC.Thebikunin-derivedpeptide (residues 1-18) was submitted to FAB-MS (positive ion). A The corresponding ions are indicated in the spectrum shown in A . molecular ion a t m/z 2413 was detected, which is 613 atomic units).Such a fragmentationpatternisconsistent witha mass units higher than that at m/z 1800 expected for the peptide. The difference is consistent with the tetrasaccharide linkage between C-6 of the terminal (reducing) N-acetylhexxylose-galactose-galactose-dehydrohexuronicacid typical of a osamine and the C-terminalAsp of the heavy chain. Similar 1-6glycosyl linkages bechondroitin AC lyase digest of Ser-linked glycosaminoglycan patterns have been observed for tween saccharide residues (17). On other analyses, a weak ion (16). The heavy chain-derivedpeptide was purified following at m/z 2839 was observed in addition to a prominent ion at chondroitin ABC lyase treatment of the cross-linked peptide m/z 2759, indicating the presence of sulfate on some GAG (see Table I, column 4a) and analyzed by negative ion FAB- moieties. Consequently, our FAB-MS data indicate that the GAG MS. Thecalculated average mass of the peptide (minus GAG) was 2019 atomic mass units. In Fig. 2 A , we show the molecular chain originatesfrom a typical xylose-galactose-galactose "inion region at m/z 2000-2900. The most abundant ion is m/ at ner core" attachment to Ser-10 of bikunin. Somewhere toward z 2759, representing an increase of 740 atomic mass units the nonreducing end of the GAG chain, an internal GalNAc from that calculated for the peptide. These results indicate is esterified, via its C-6 hydroxyl, to the C-terminal Asp of the presence of a tetrasaccharide composed of 2 N-acetylhex- HC3. These dataprovide direct evidence for a covalent interlink between the two protein osamines, 1 hexuronic acid, and 1 dehydrohexuronic acid on chainchondroitin4-sulfate the peptide. Examination of the fragment ionsallowed eluci- chains of PaI. dation of the GAG tetrasaccharide. Oligosaccharides undergo GalNAc Is Esterified to a-Carboxylate of C-terminal Aspfragmentation in FAB primarily at the0-glycosyl bond with Since the siteof the chondroitin 4-sulfate attachmentis a Chydrogen transfer, asshown in Fig. 2A (inset). This fragmen- terminal Asp, FAB-MS analysis did not allow us to ascertain tation pattern is evident in the spectra. Other fragment ions whether the GalNAc was esterified to the a- or P-carboxylate are also observed at m/z 2741 (-HzO) and m/z 2715 and 2336 of the C-terminal Asp. The position of attachment to theC(-COz) and from fragmentation of the glycosyl ring (Fig. 2B). terminal Asp of HC3 was determined following modification The fragment ions at m/z 2034, 2064, 2094, and 2124 result of carboxylate groups by glycinamide in the presence of EDC. This method is based on the assumption that one of the fromsequential losses of formaldehyde (-30 atomicmass c.olI*--H(--c"--c

"OCYz

750

Glycosaminoglycan-mediated Novel Cross-link Protein

carboxylates of the C-terminal Aspwill be occupied in an ester to GalNAc, but that the other will be free to react with nucleophiles in the presence of EDC. In this case, we would expect to promote amide formation between available carboxylate groups of the peptide and theamino group of glycinamide. Therefore, the carboxylate group of Asp-17 that is esterified to GalNAc is identified by its lack of reactivity with glycinamide. Glycinamide was allowed to react with 5 nmol of the Cterminal peptide (see Table I, column 4a). Excess reagent was removed, and the modified peptide was deglycosylated with 50 mM NaOH, purified by RP-HPLC, and submitted amino to acid analysis. Compositional analysis revealed the presence of 3 Gly residues. Since the peptide only contains 1 Gly, this indicated that two carboxylates had been modified by glycinamide. These are likely the &carboxylate of Asp-15 and one of the carboxylate groups of Asp-17. Onthe other hand,if the C-terminal peptide was deglycosylatedby using 50 mM NaOH before modification with glycinamide, 4 Gly residues were detected by compositional analysis, indicating that an additional carboxylate had been modified, presumably the carboxylate of the Asp uncovered bydeglycosylation. When the latter material was submitted to peptide sequence analysis, PTH-8-Asp-glycinamide was identified in cycle 17and PTHGly in cycle 18. This is to be expected since both carboxylates of Asp-17 were modified. However,peptide sequence analysis of material that had been modified with glycinamide before deglycosylation showed PTH-P-Asp-glycinamide in cycle 17, but no PTH-Gly above background in cycle 18. This key observation indicates that thea-carboxylate of the C-terminal peptide (see Table I, column 4a) is unavailable for modification and is likely the site of attachment of the GalNAc unit. Therefore, we conclude that the GalNAc is esterified to the a-carboxylate of Asp-17, which is the C-terminal residue of HC3.

size since chondroitin sulfate chains attached to proteins are usually heterogeneous (18). Our data did not allow us to ascertain whichGalNAc is attached tothe Asp of HC3; indeed, the site of attachment may vary frommolecule to molecule depending on the mechanism responsible for the attachment. Whereas this is probably the first description of a covalent GAG protein cross-link, other types of carbohydrate crosslinks have been found in connective tissue proteins. It has recently become apparent that the aging process results in nonenzymatic glycation (nonenzymatic glycosylation) of certain proteins that results in cross-linking of collagen (19) and other connective tissue proteins(20).These “senescence” cross-links are mediated by sugars, usually pentoses, and are thought to be responsible for the refractivity of aged tissues to solubilization or digestion. Senescence carbohydrate crosslinks are rathernonselective and take several days to form in vitro and probably months to years in vivo (19). By contrast, the GAG-mediated cross-linking of Pa1 occurs in less than 1 h following synthesis of the constituent protein chains in cultured cells (21). Several mechanisms are possible for the synthesis of the 0-glycosidic link between GalNAc and Asp, and the chemical process likely results from condensation of a carboxylic acid with an alcohol since the product is an ester. Whereas we do not know the order of reaction of the protein chains with the GAG chain, it is reasonable to suggest that GAG is first added to Ser-10 of bikunin since glycosylation of this type normally occurs early in protein processing within the endoplasmic reticulum (22). The next step in cross-linking would occur following condensation of the C-6 hydroxyl of a GalNAc with the a-carboxylate of the HC3 Asp. An alternative possibility for biosynthesis of the cross-link is suggested by the glycosylphosphatidylinositol modification of the Cterminal residue of several proteins that anchors these tocell membranes (23). Theseso-called glycosylphosphatidylinositol anchors react to displace small C-terminal peptides of the precursor proteins, becoming linked to the a-carboxylate of the new C-terminal residue. However, in these cases, attachment is by nucleophilic displacement mediated by the amino group of the glycosylphosphatidylinositol ethanolamine moiety and results in an amide link. In characterizing the mechanism responsible for formation of the GalNAc-Asp ester, it should not be overlooked that HC3 or the 27-kDa putative C-terminal polypeptide of the HC3 precursor may perform this function. In this case, the reaction would not necessarily be catalytic since HC3 may fold intoa form that activates the Asp and recognizes a GalNAc of the chondroitin 4-sulfate chain. It would then be,

DISCUSSION

Our data are consistentwith the structure shown in Fig. 3. We were unable to detect the presence of al-microglobulin of the C-terminal peptide of HC3. Since the C-terminal Asp or HC3 is a-linked to thechondroitin 4-sulfate chain, the putative C-terminal peptide extension of the precusor must have been removed during biosynthesis. Similarly, the N-terminal region of the bikunin precursor (a,-microglobulin) must also have been removed before secretion of Pa1 into the blood. The chondroitin 4-sulfate chain that originates from Ser-10 b = 16 of bikunin is shown, for simplicity, to contain a disaccharide units (Fig. 3). We suspect that this is an average

+

Gal I

I

- [GlcUA- GalNAc] - GlcUA- GalNAc-[GlcUA- GalNAc] a

FIG. 3. Cross-linkingof PaI. The heavy chain of Pa1 is known as HC3 to distinguish it from the homologous heavy chains of I d . The light chain, common to Pa1 and I d , is called bikunin to signify its tandem Kunitz domain structure (6). A chondroitin 4-sulfate chain originates from Ser-10 of bikunin in a standard GAG linkage. Somewhere toward the nonreducing end of this chain, a GalNAc unit is esterified, via its C-6 hydroxyl, to the acarboxyl of the C-terminal Asp of the heavy chain. The bold open boxes represent the protein chains of Pa1 and are to scale. The other boxes represent components of the precursors ofbikunin and HC3, cul-microglobulin(alrn), and the C-terminal polypeptide, respectively, that are not found in mature PaI. GlcUA, glucuronic acid.

Novel Glycosaminoglycan-mediated Protein Cross-link

751

in essence, an enzyme that turns over only once. Ananalogy 2. Partridge, s. M. (1962) Protein Chem. 17-227-302 is the reaction ofthe a2-macroglobu]in thio] ester with amines, 3- Eyre, D. Paz, M. and Gallop, p. M. (1984) Annu. Reu. Biochem. 53, 717-748 a reaction with properties of a single turnover of a transglu4. Enghild, J. J., Thbgersen, I. B., Pizzo, S. V., and Salvesen, G. taminase (24). (1989) J. Biol. Chem. 264, 15975-15981 AreThere Other Glycosaminoglycan-cross-linkedPro5. Diarra-Mehrpour, M., Bourguignon, J., Sesbohe, R., Mattel,M.&ins?-We have considered whether the protein-glycosamiG., Passage, E., Salier, J.-P., and Martin, J.-P. (1989) Eur. J . noglycan-protein (PCP) covalent adductof Pa1 is likely to be Biochem. 179,147-154 6. Gebhard, W., Schreitmuller, T., Hochstrasser, K., and Wachter, found in other proteins. We have shown (4) that the human E. (1989) Eur. J. Biochem. 181, 571-576 blood protein I d is an assembly of the bikunin chain with 7. Gebhard, W., and Hochstrasser, K. (1986) in Proteinase hhibitwo distinct heavy chains, designated HC1 and HC2. This protein complex shares many properties with PaI. The heavy tors (Barrett, A. J., and Salvesen, G., eds) pp. 389-401, Elsevier/ North-Holland Biomedical Press, Amsterdam chains are attached by a chondroitin-like chain to the light 8. Bury, A. F. (1981)J. Chromatogr. 213, 491-500 chain that is apparently identical to the Pa1 bikunin (4, 25). 9. Uriel, J., and Berges, J, (1968) Nature 218, 578-580 The chondroitin-like chain originates from Ser-10 of bikunin; Matsudaira, p. (1987) J . ~ ~them, ~ 262, l ,1o035-10038 but, whereas it links to a single heavy chain in (HC3), in 11. Enghild, J. J., Thdgersen, I. B.,Roche, P. A., and Pizzo, S. V. I d it links totwo heavy chains (HC1 andHC2). HC1 shows (1989) Biochemistry 28, 1407-1412 -52% identity and HC2 -37% identity to theregion of HC3 12. Meltzer, N. M., Taus, G. I., Gruber, S., and Stein, S. (1987) Anal. Biochem. 160,356-361 so far sequenced (5,6). Moreover, the precursors of HC1 and HC2 also contain 27-kDa putative C-terminal extensions that 13. Means, G. E., and Feeney, R. E. (1971) Chemical Modification of Proteins, pp. 139-148, Ho1den:Day Inc., San Francisco are closely related to thatof the HC3precursor. Finally, HC1 and HC2 precursors conform to the sequence motif ~ ~ ~ 14. - Hochstrasser, p ~ ~ K., -Schonberger, 0. L., Rossmanith, I.. and W a d ter, E. (1981) Hoppe-SeylerS 2. Physiol.Chem. 362, 1357His-Phe-Ile-Ile downstreamof the GalNAc-Asp esterification 1362 site in Pal. we predict that Ial contain the 15. Kaumeyer, J. F., Polazzi, J, O., and Kotick, M. p, (1986) Nucleic P G P cross-link with, interestingly, two distinct heavy chains Acids Res. 14, 7839-7850 attached to theGAG chain. 16. Yamagata, T., Saito, H., Habuchi, O., and Suzuki, S. (1968) J . Thepresence of thePGPcross-link in otherproteins is Biol. Chem. 243, 1523-1535 presently unknown. There may be a certain feature of the 17. Biermann, C. J. (1988) Adu. Carbohydr. Chem.Biochem. 46,251271 donor GAG chain or the acceptor heavy chains that forbids interactions with other proteins. In the caseof Pa1 and I d , 18. Carney, S. L. (1986) in Carbohydrate Analysis: A Practical APproach (Chaplin, M. F., and Kennedy, J. F., eds) pp. 97-141, the majority of chains are isolated in the intact cross-linked IRL Press, Oxford complexes (4). However, during our studies, we have noticed s.,Avigad, G., Eikenberry, E. F., and Brodsky, B. (1988) that thelight chainwith theGAG chain attachedstainspoorly 19. Tanaka, J. Biol. Chem. 263, 17650-17657 with Coomassie (Fig* 3, and that and Ial 20. Sell, D. R., and Monnier,V. M. (1989) J. Biol. Chem. 264,21597and chondroitin 4-sulfate-containing fragments thereof are 21602 extremely heterogeneous on ion-exchange and reverse-phase 21. Bourguignon, J., Sesboue, R., Diarra-Mehrpour, M., Daveau, M., chromatography media. Therefore, it is possible that small and Martin, J.-P. (1989) Biochem. J . 261, 305-308 portions O f known proteins containing PGP covalent cross- 22. Lindahl, U., and Hook, M. (1978) Annu. Reu. Biochem. 47, 385417 links have been overlooked. A.7

1A9

Acknowledgments-We thank Pat BurksandAndreaTillotson for preparing the manuscript, B. Fraser-Reid for helpful discussions, and B. Caterson for suggesting the use of the monoclonal antibodies. REFERENCES 1. Folk, J. E., and Finlayson, J. S. (1977) Adu.ProteinChem. 1-133

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