Proc. Natl. Acad. Sci. USAVol. 92, pp. 7367-7371, August 1995Biochemistry

Carbohydrate gluing, an architectural mechanism in thesupramolecular structure of an annelid giant hemoglobin

(lectin/linkers)

SATOSHI EBINA*t, KEIKO MATSUBARA*, KuNIAKI NAGAYAMA*, MARIKO YAMAKI*, AND ToSHIO GOTOHt*Nagayama Protein Array Project, Exploratory Research for Advanced Technology, Research Development Corporation of Japan, 5-9-1 Tokodai, Tsukuba, 300-26Japan; and tDepartment of Life Science, Faculty of Integrated Arts and Sciences, University of Tokushima, Tokushima, 770 Japan

Communicated by John T. Edsall, Harvard University, Cambridge, MA, May 1, 1995 (received for review February 10, 1995)

ABSTRACT We report a carbohydrate-dependent su-pramolecular architecture in the extracellular giant hemoglo-bin (Hb) from the marine worm Perinereis aibuhitensis; we callthis architectural mechanism carbohydrate gluing. This studyis an extension of our accidental discovery of deterioration inthe form of the Hb caused by a high concentration of glucose.The giant Hbs of annelids are natural supramolecules con-sisting of about 200 polypeptide chains that associate to forma double-layered hexagonal structure. This Hb has 0.5% (wt)carbohydrates, including mannose, xylose, fucose, galactose,glucose, N-acetylglucosamine (GlcNAc), and N-acetylgalac-tosamine (GalNAc). Using carbohydrate-staining assays, inconjunction with two-dimensional polyacrylamide gel electro-phoresis, we found that two types of linker chains (LI and L2;the nomenclature of the Hb subunits followed that for anothermarine worm, Tylorrhynchus heterochaetus) contained carbo-hydrates with both GIcNAc and GalNAc. Furthermore, twotypes of globins (a and A) have only GlcNAc-containingcarbohydrates, whereas the other types of globins (b and B)had no carbohydrates. Monosaccharides including mannose,fucose, glucose, galactose, GlcNAc, and GalNAc reversiblydissociated the intact form of the Hb, but the removal ofcarbohydrate with N-glycanase resulted in irreversible disso-ciation. These results show that carbohydrate acts nonco-valently to glue together the components to yield the completequaternary supramolecular structure of the giant Hb. Wesuggest that this carbohydrate gluing may be mediatedthrough lectin-like carbohydrate-binding by the associatedstructural chains ("linkers").

(Fig. 1B). This surprising finding, together with our subsequentsystematic studies using Hb from another type of marineworm, Perinereis aibuhitensis Hb (5), described in this report,has led us to a better understanding of the critical role ofcarbohydrates in the supramolecular structure of giant Hbs.The giant Hbs are very large proteins with disk-shaped

structures that are about 20 nm thick and 30 nm in diameter.They contain 12 similar units (called submultiples) that forma double layer of two sets of six hexagonally arranged submul-tiples (6). This structure can be seen in the TEM image shownin Fig. IA. Biochemical studies have shown that each of thesubmultiples contains four types of globin chains, all of whichcontain heme (7-11) [called a, A, b, and B (11)], and at leasttwo types of polypeptide chains that contain no or little heme(12-14). These non-heme chains are thought to link the 12submultiples, thus holding the entire molecule together (12,13). These chains are called linkers and are designated Li andL2 (14, 15). Because the entire molecule consists of about 200polypeptide chains (13, 14, 16), some of which contain carbo-hydrates (15, 17, 18), it is called a supramolecule. The mech-anism by which linkers mediate the assembly of the manypolypeptide chains to yield the double-layered hexagonal formis not yet understood. We focus here on the essential role ofcarbohydrate in forming the supramolecular architecture. Inthis report, the nomenclature of the subunits, a, A, b, B, Li,and L2, followed that for another marine worm, Tylorrhynchusheterochaetus (11, 14), although it may not be applied to allgiant Hbs.

A recent breakthrough in science is the supramolecular fab-rication of large complexes from small molecules (1). Theuniqueness of this fabrication, as compared with the conven-tional fabrication (such as polymer synthesis), is that thefabrication of large structures is based on "self-assembly" ofsmall compounds by extensive use of noncovalent bonds. Thisstrategy may model the first evolutionary step from proteins tosupramolecules in the biological hierarchy: proteins -- su-pramolecules -k organelles -- cells -- tissues/organs -* indi-viduals -- societies. This research field is thus called supramo-lecular chemistry (2). Understanding the mechanisms that rulebiological hierarchies is a challenging research theme in manyscientific fields. Knowledge currently available on biologicalhierarchy, even for the evolutionary step from proteins tosupramolecules is, however, limited.

In our attempts to fabricate two-dimensional protein arrayson a mercury surface from proteins and protein complexes inhigh concentrations of glucose solutions (0.55 M) (3, 4), weserendipitously discovered (Fig. 1) that glucose distorted theintact form of giant Hb from the marine polychaete Marphysasanguinea (Montagu), resulting in swelling of the Hb molecules

MATERIALS AND METHODSGiant Hb. The giant Hb was purified from a homogenate of

the marine worm Perinereis aibuhitensis by the sequentialprocedures of centrifugation, ammonium sulfate fractionation,gel filtration, and ultracentrifugation, as detailed earlier (5).

Dissociation-Reassociation ofHb with Monosaccharides.Asolution of the giant Hb (30 ,ul, 0.1 mg/ml) in 50 mMphosphate buffer (pH 7.2) was incubated overnight at 25°Cwith various concentrations of monosaccharides, such as man-nose, fucose, glucose, galactose, N-acetylglucosamine (Glc-NAc), and N-acetylgalactosamine (GalNAc). We obtained thedissociation curve by determining the intact form of the Hb infive different areas (30 x 30 ,um) of electron microscope gridsof each sample. To study the reassociation, we further dialyzedthese solutions overnight at 4°C against the same buffer butwith no monosaccharides. We also studied the dissociation andreassociation using gel filtration with a Superose 6 column(Pharmacia) and an elution buffer of 50 mM phosphate (pH7.2).

Deglycosylation with N-Glycanase. After the initial incuba-tion with monosaccharides, the solution was incubated at 37°C

Abbreviations: TEM, transmission electron microscope (microscopy);WGA, wheat germ agglutinin; DBA, Dolichos biflorus agglutinin.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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FIG. 1. Transmission electron microscope (TEM) images of giant Hb from Marphysa sanguinea (Montagu) without (A) and with (B) glucoseat 0.55 M in 50 mM phosphate buffer (pH 7.2). The Hb molecules swelled with glucose. (Bar = 100 nm.)

for 24 hr with 0.1 M GalNAc and 1 mM protease inhibitor(phenylmethylsulfonyl fluoride) in the presence of 3 units ofN-glycanase (Flavobacterium meningosepticum obtained fromGenzyme) and then dialyzed overnight at 4°C.TEM. A drop of each sample solution of giant Hb at 0.01

mg/ml in 50 mM phosphate buffer (pH 7.2) was placed on acarbon film, which was mounted on a copper grid used forTEM analysis and sputtered by using an ion-sputter (JFC-1100E, JEOL). After the negative staining of the sample with2% uranyl acetate, we determined the intact form of the giantHb using a TEM (EM1200EXII, JEOL) (19).

Carbohydrate-Staining Assay with Lectins. We determinedthe carbohydrates in the subunits by using lectins (20), such aswheat germ agglutinin (WGA) and Dolichos biflorus agglutinin(DBA), in conjunction with two-dimensional PAGE in thepresence of 8 M urea, in which the first- and second-dimensionruns were made with isoelectric focusing PAGE in the pHrange of 3.0-7.0 and SDS/PAGE at pH 7.0, respectively (21).To modify the sulfhydryl groups, we first reduced the proteinsample with 1% mercaptoethanol in 8 M urea at pH 7.2 andthen treated it with 4-vinylpyridine at pH 7.2 (22). We iden-tified the subunits on the two-dimensional gel according to theidentification for Tylorrhynchus heterochaetus (11, 14).

RESULTSWe first analyzed the carbohydrates contained in the whole Hbmolecule by using the fluorescent postlabeling method fol-lowed by hydrolysis and identification of the monosaccharidesusing HPLC separation (23). We found 0.5% (wt) carbohy-drates in the Hb. We identified the composition of thecarbohydrates as follows: mannose, 22%; xylose, 7%; fucose,5%; galactose, 3%; glucose, 55%; GlcNAc, 6%; and GalNAc,2%. The high content of glucose probably involved a contam-

inant from the gel matrix in the gel filtration of giant Hb. Sialicacid was not identified. Next, by using the carbohydrate-staining assay technique with lectins (WGA and DBA) inconjunction with 2D PAGE, we confirmed that Li and L2 werestrongly positive to WGA and DBA staining. These findings ofthe presence of carbohydrates are consistent with the report in1973 of the presence of GalNAc and mannose in the molecule(17) and the recent report of a significant amount of carbo-hydrates in Li of earthworm Hb (Lumbricus terrestris) (15, 18).Furthermore, we found that globins a and A were slightlypositive only to the WGA staining, and b and B were negativeto both stainings. Fig. 2 shows our results for the carbohydrate-staining assay using WGA and DBA.Based on the presence of these carbohydrates, we next

studied the morphological changes associated with dissociationof giant Hb by incubating it with several types of monosac-charides (mannose, fucose, glucose, galactose, GlcNAc, andGalNAc) in 50mM phosphate buffer (pH 7.2). In the presenceof each of these monosaccharides, the intact form of giant Hbwas dissociated into smaller units, which we identified usingTEM and the gel filtration technique. We confirmed withthese experiments the relatively strong dissociating effect ofGalNAc, which was followed in strength by mannose. Fig. 3shows the TEM images of the giant Hb in the presence ofdifferent concentrations of GalNAc (Fig. 3A), a curve of thedissociation as a function of GalNAc (Fig. 3B), and the elutionpatterns of gel filtration for some of these Hb samples (Fig.3C). The Hb that had been dissociated in the presence of lowconcentrations of GalNAc was characterized by different-sizedparticles (as seen in image A2), where some particles hadstructures similar to that of the intact form but lacked the finestructures that are clearly seen in the intact form (image Al).TEM identified this type of morphological change due to lowconcentrations of GalNAc, whereas the gel filtration did not

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kDa

97.466.342.430.0

20.1

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pH pH 7 3 pH

FIG. 2. Carbohydrate staining assay of components in the giant hemoglobin (Hb) from Perinereis aibuhitensis in conjunction with two-dimensional PAGE. The Hb was reduced with mercaptoethanol and then modified with 4-vinylpyridine before subjecting the sample to theelectrophoresis. The first dimension of PAGE was isoelectric focusing (pH range, 3.0-7.0) and the second-dimension run was SDS/PAGE at pH7.0. (A) Components thus separated and stained with Coomassie brilliant blue. (B) Those with WGA staining. (C) Those with DBA staining.

discriminate such a small change. It was seen that the disso-ciated Hb tended to adsorb to the matrix of the gel filtration.With higher concentrations of GalNAc, the Hb was furtherdissociated into smaller particles, resulting in extensive for-mation of aggregates as typically seen in image A3. Therefore,we obtained the dissociation curve, which is given in Fig. 3B,by counting the intact form in the TEM images, with themidpoint for dissociation being about 10 mM GalNAc, al-

A

though the dissociation did not thus likely follow a simplecurve. By dialyzing a solution from A3 in the phosphate bufferthat contained no monosaccharides, we discovered the exten-sive reassociation to the double-layered hexagons of the intactHb as seen in image A4. The extent of reassociation was >70%of that from the incubation at 25°C (image Al) with the sameincubation at 37°C for 24 hr but without N-glycanase. We thenstudied the effect of deglycosylation on reassociation of giant

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Elution Time (min)

FIG. 3. (A) TEM images of giant Hb from Perinereis aibuhitensis in the presence of different concentrations of GalNAc. (Bar = 100 nm.) (B)Dissociation curve of the giant Hb, as a function of the concentration of GalNAc, that was obtained by counting the intact Hb molecules fromthe TEM images. (C) Elution patterns in the gel filtration. InA, a solution of giant Hb in 50mM phosphate buffer (pH 7.2) was incubated overnightwithout (image Al, the intact Hb) and with various concentrations of GalNAc at 25°C (image A2, with 10 mM GalNAc; image A3, with 100 mMGalNAc). In image A2, the Hb is seen as different-sized particles; some particles hold structures similar to that of the intact form but lack the finestructure (arrows). In image A3, the Hb was further dissociated into smaller particles, resulting in formation of aggregates. A solution from imageA3 was dialyzed overnight at 4°C in the same buffer but with no GalNAc, resulting in extensive recovery of the intact form (typically indicatedby the arrows in image A4). Deglycosylation by incubating a solution from A3 at 37°C for 24 hr with N-glycanase inhibited the reassociation (imageA5). In the gel filtration patterns (C), the recovered Hb by the dialysis (image A4) is seen at the same position for that of the intact form (imageAl), whereas the deglycosylated Hb (image A5) was eluted at sizes between 67 and 14 kDa.

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standard(kDa) 66944067 14A4,4 4 4,

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Hb using N-glycanase. The cleaved monosaccharides weresubsequently removed by dialysis against the same bufferwithout monosaccharides. Image A5 shows that the reassocia-tion (seen in image A4) was completely inhibited by deglyco-sylation. The amount of deglycosylation obtained with N-glycanase was not noticeably affected by the presence of either0.1% SDS or 5 M urea in the incubation medium. Thecarbohydrate-staining assay revealed that the deglycosylationdecreased the apparent molecular mass of Li and L2 by about10% and 30%, respectively, due to liberation of carbohydrates.The elution patterns in the gel filtration (Fig. 3C) show thesame dissociation-reassociation profile and the irreversibledissociation due to the deglycosylation.We also noticed in these experiments that type I and II

monosaccharides (24) (fucose, galactose, and GalNAc) re-sulted in the formation of aggregates, whereas type IIImonosaccharides (24) (mannose, glucose, and GlcNAc) didnot. It should be noted that this typing of monosaccharides wasbased on the specificity of the inhibitory activity of themonosaccharides toward agglutination of red blood cells in thepresence of lectin (24). The difference spectra in the UV ofHbin the presence and absence of mannose at concentrationsranging from 0.01 to 0.5M in 50mM phosphate buffer (pH 7.2)suggested that the dissociated Hb retained most of the sec-ondary and tertiary structures of the intact form.

DISCUSSIONWe have obtained the following experimental results in thisstudy of the giant Hb: (i) the intact molecule contains GlcNAc,GalNAc, and several types of hexose, but no sialic acid; (ii)WGA binds the carbohydrates in Li, L2, a, and A, whereasDBA binds only Li and L2; (iii) GalNAc and mannose stronglyand reversibly dissociated the intact form; (iv) reassociation isinhibited by deglycosylation; (v) dissociation preserves most ofthe secondary and tertiary structures; and (vi) type I/IImonosaccharides produced aggregates, whereas type III didnot. Results i and ii show that Li and L2 possess two types ofcarbohydrates-namely, GlcNAc-containing and GalNAc-containing carbohydrates-while a and A have only GlcNAc-containing carbohydrates. Result iii shows that the carbohy-drates must be bonded by a subunit that has a high affinity toGalNAc (the midpoint for the dissociation was about 10 mMGalNAc, as seen in Fig. 3) and also to mannose. Therefore,with result iv, we conclude that intramolecular carbohydratesare responsible for gluing the subunits together to yield theintact form, because this gluing cannot be attributed only toprotein-protein interactions. The most probable candidatesfor these intramolecular carbohydrate interactions are thosecovalently bound to the linkers. Based on result v, the gluingis mainly a process of quaternary assembly. Supramolecules innature contain various types of complexes, such as proteincomplexes [e.g., proteasome, which is a multicatalytic protein-

ase complex (25)] and protein-nucleic acid complexes (e.g.,ribosome). Protein-lipid complexes [e.g., photoreceptorG-protein (26)] occur as heterooligomers, which are not evensupramolecules. However, there has been no report of pro-tein-carbohydrate-dependent supramolecules, even thoughhomotetrameric assembly and subsequent aggregation of So-phora japonica lectin were reported to be mediated by in-tramolecular carbohydrates (27). In this work, we demonstratethat carbohydrates noncovalently glue together components ofthe giant Hb to yield the complete quaternary supramolecularstructure. We propose to call this supramolecular architecturalmechanism carbohydrate gluing.

Studies of sequence similarities between linker chains andlectins show the possible relevance of lectin-type binding in thegluing as follows. Linker chains are multifunctional proteinsthat consist of three distinct segments (18). Our recent studyof Perinereis Hb has shown that the linkers are highly homol-ogous to those in another marine worm, Tylorrhynchus hetero-chaetus Hb (7, 14). First, we noticed that cysteine-rich motifs(14, 18) in segment 2 of Tylorrhynchus Li and L2, earthwormLumbricus terrestris Li (18), and marine worm Neanthesdiversicolor L2 (28) have significant similarities with each of thefour-repeated units in WGA (29). Even more significantly,Asp-29 is known to participate in forming a binding site inWGA for GlcNAc-containing carbohydrates (29). Fig. 4 sum-marizes these sequence alignments. As seen in Fig. 4, there area total of eight gaps in the alignments of Tylorrhynchus L2 andWGA. Nevertheless, the correspondence between these se-quences is suggestive of a possible WGA-like function of theL2.

Carbohydrate staining with DBA shows the presence ofGalNAc-containing carbohydrates in Perinereis Li and L2.Interestingly, segment 3 in Tylorrhynchus Li (but not in L2)contains short sequences (namely, Val-Pro and Pro-Asp---Gly-Gly) that are also seen in a galactose-binding family ofC-type lectins (30), although these peptide sequences may betoo short to be meaningful. It is important to note that thereis a similar connectivity in two sets of disulfide bridges betweensegment 3 (namely, Cys-151-Cys-249 and Cys-226-Cys-236) inTylorrhynchus Li and L2, Neanthes L2, and Lumbricus Li andin the carbohydrate-binding domain in C-type lectins, aspreviously reported (28). Therefore, there might be two typesof binding pairs: (i) segment 2 with GlcNAc-containing car-bohydrates and (ii) segment 3 with GalNAc-containing car-bohydrates. In this respect, the formation of two types ofdissociation processes due to the two types of monosaccharidesthat coincide with Makela's classification (24) (result vi in theabove) may support the presence of the two types of bindingpairs described above. Suzuki and Riggs (15) suggested thatthe cysteine-rich motif, which is also present in other types ofproteins, such as low density lipoprotein receptor (31), com-plement C9 (32), and renal glycoprotein GP330 (33), isresponsible for binding with other protein molecules, in which

segment 2 ISISs SIS IsSIS $SS Is g s gMIMI SIMM;HISSII

Vylorrhynchus Li HHCDDDHLS-CKI-C-DVAFTCIGHNLVCD- -GHKDCLNGHDEDEETCSLumbrlcus Li HHCDEHESE-CR--GDVPECIHDLLFCD--GEKDCRDGSDEDPETCSNoanthes L2 NGCDARHYQ-CG--GNTPYCIADTLVCD- -GSPDCPNGSDESEDICH

Tyloarr2hy2chus L2 NGCEPRHFaG -, IBLTiD- -4PD DSDVCR4I1 Q>COEQ PNNLC~-CSQYG- - 1QYCIGKCDGACSTS

WGA 44 KRCGSQAGGATCPNNHC-CSQYGH- -CGFGAEYCGAGCQGGPCRAD87 IKCGSQSGGKLCPNNLC-CSQWGY--CGLGSEFCGGGCQSGACSTD130 KPCGGDAGGRVCTNNYC-CSKGGS--CGIGPGYCGAGCQSGGCDA

FIG. 4. Alignment of segment 2 in linkers of the giant Hb from Tylorrhynchus heterochaetus (7, 14), Lumbricus terrestris (18), and Neanthesdiversicolor (28) with the repeated sequences in WGA (29). Cys-3, Cys-12, Cys-18, Cys-24, Cys-35, and Asp-29 (numbers for the first sequence inWGA; Asp-29 in WGA is involved in binding the carbohydrates) are aligned. The shaded letters indicate homologous amino acids in TylorrhynchusL2 with the first sequence in WGA.

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binding is probably based on both multielectrostatic andnonpolar interactions. We thus propose that carbohydrategluing, very probably in collaboration with these interactions,participates in constructing the supramolecular structure ofthe giant Hb.As discussed above, we suggest that carbohydrate gluing in

the giant Hb relies on two types of lectin-carbohydrate bind-ings. This in turn leads us to speculate that a skeleton-likestructure made of two types of linkers (Li and L2) anchorscarbohydrates of a and A in submultiples to yield the double-layered hexagonal form. This speculation can be considered asan extension of the bracelet model originally proposed byVinogradov et al. (12). Therefore, the x-ray crystallography ofLumbricus Hb, which is currently in progress (34), is of greatinterest.Nature uses carbohydrates extensively as key substances in

many cell processes, such as differentiation and morphogenesis(see, for example, ref. 35). In this article, we provide insightinto the key role of carbohydrates in the evolution fromproteins to supramolecules in the biological hierarchy.

We thank Mr. Akihiro Shibuya (University of Tokushima) forproviding the purified Perinereis giant Hb and Ms. Tomoko Tange (thisproject) for providing the analyzed sequence homology. We also thankDr. Tamao Endoh (University of Tokyo) and Dr. Jun Hirabayashi(Teikyo University) for their fruitful discussions. We are indebted toProf. Pauline M. Harrison (University of Sheffield) for her continuousencouragement of this research. Finally, we thank Dr. Robert Lewis(Tsukuba Research Consortium) for his critique of this manuscript.

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