Báo cáo khoa học: Comparative analysis of carbohydrate-binding properties of two tandem repeat-type Jacalin-related lectins, Castanea crenata agglutinin and Cycas revoluta leaf lectin docx

Obvi-ously, both CCA and CRLL showed affinity for a wide range of N-linked glycans, but not at all for glycolipid-type glycans, which lack mannose.. CRLL also showed significant affinity fo

of two tandem repeat-type Jacalin-related lectins,

Castanea crenata agglutinin and Cycas revoluta leaf lectin Sachiko Nakamura1, Fumio Yagi2, Kiichiro Totani3, Yukishige Ito3and Jun Hirabayashi1

1 Glycostructure Analysis Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Japan

2 Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Japan

3 RIKEN (The Institute of Physical and Chemical Research), Saitama, Japan

Lectins are carbohydrate-binding proteins distributed

in all of the organisms characterized so far A large

number of plant lectins have been isolated and

charac-terized [1,2] Van Damme et al classified them into

seven families based on their molecular structures and

carbohydrate-binding specificities [3] Members

belong-ing to each family share some common properties

Compared with legume and monocot lectin families,

jacalin-related lectins (JRLs) were originally assumed

to form a relatively small family [4] During the last decade, however, many new members belonging to this family were discovered, and some novel features became evident As a result, JRLs can now be classi-fied into two subgroups in terms of their carbohydrate binding specificities, i.e galactose-binding-type JRLs (gJRLs) and mannose-binding-type JRLs (mJRLs) [5]

Keywords

carbohydrate binding specificity; frontal

affinity chromatography; Jacalin-related

lectin family; lectin

Correspondence

J Hirabayashi, Glycostructure Analysis

Team, Research Center for Glycoscience,

National Institute of Advanced Industrial

Science and Technology, AIST Tsukuba

Central 2, 1-1-1, Umezono, Tsukuba,

Ibaraki 305-8568, Japan

Fax: +81 29 861 3125

Tel: +81 29 861 3124

E-mail: jun-hirabayashi@aist.go.jp

(Received 27 December 2004, revised 23

February 2005, accepted 4 April 2005)

doi:10.1111/j.1742-4658.2005.04698.x

Lectins belonging to the jacalin-related lectin family are distributed widely

in the plant kingdom Recently, two mannose-specific lectins having tan-dem repeat-type structures were discovered in Castanea crenata (angio-sperm) and Cycas revoluta (gymno(angio-sperm) The occurrence of such similar molecules in taxonomically less related plants suggests their importance in the plant body To obtain clues to understand their physiological roles, we performed detailed analysis of their sugar-binding specificity For this purpose, we compared the dissociation constants (Kd) of Castanea crenata agglutinin (CCA) and Cycas revoluta leaf lectin (CRLL) by using 102 pyridylaminated and 13 p-nitrophenyl oligosaccharides with a recently developed automated system for frontal affinity chromatography As a result, we found that the basic carbohydrate-binding properties of CCA and CRLL were similar, but differed in their preference for larger N-linked glycans (e.g Man7–9 glycans) While the affinity of CCA decreased with

an increase in the number of extended a1–2 mannose residues, CRLL could recognize these Man7–9 glycans with much enhanced affinity Nota-bly, both lectins also preserved considerable affinity for mono-antennary, complex type N-linked glycans, though the specificity was much broader for CCA The information obtained here should be helpful for understand-ing their functions in vivo as well as for development of useful probes for animal cells This is the first systematic approach to elucidate the fine spe-cificities of plant lectins by means of high-throughput, automated frontal affinity chromatography

Abbreviations

CCA, Castanea crenata agglutinin; CRLL, lectin from leaves of Cycas revoluta; CRD, carbohydrate-recognition domain; FAC, frontal affinity chromatography; gJRLs, galactose-binding-type related lectins; JRLs, related lectins; mJRLs, mannose-binding-type Jacalin-related lectins; M2M2M3Mb, Mana1–2Mana1–2Mana1–3Manb; M2M3M6Mb, Mana1–2Mana1–3Mana1–6Manb; MTX, methotrexate; M3GN2, Man3GlcNAc2; PA, pyridylaminated; pNP, p-nitrophenyl.

gJRLs are represented by Jacalin, known as a useful

probe for IgA [6], and also by Maclura pomifera

agglu-tinin [4] and Morus nigra aggluagglu-tinin (Morniga G) [3]

All of these lectins, which come from Moraceae plants,

have common structural features: they are composed of

four protomers consisting of a short b-chain (2 kDa)

and a long a-chain (13 kDa) as a result of proteolytic

cleavage of a precursor polypeptide [3,7] On the other

hand, the mJRL subgroup comprises many more

mem-bers, such as Calsepa isolated from Calystegia sepium

[8], Artocarpin from Artocarpus integrifolia [9], Heltuba

from Helianthus tuberosus [10], BanLec from Musa

acu-minate [11], Morniga M from M nigra [7], Orysata

from Oryza sativa [12,13] and PAL from Phlebodium

aureum [14] Although these mJRLs were proved to

form a b-prism fold I structure consisting of one

carbo-hydrate-recognition domain (CRD) similar to Jacalin

[15–17], no proteolytic modification of mJRLs occurs

There is a view that the lack of proteolysis of them

may result in preservation of their mannose⁄ glucose

specificity [18] Although all of the mJRLs mentioned

above are of the single CRD type (15–16 kDa), new

members of much larger molecular size (
33 kDa)

were discovered recently from Castanea crenata

(angio-sperm) and Cycas revoluta (gymno(angio-sperm) [19,20]

Sequence analysis of these large mJRLs, i.e C crenata

agglutinin (CCA) and C revoluta leave lectin (CRLL)

revealed that they form a tandem-repeat structure

composed of two jacalin-type CRDs [21,22] Therefore,

mJRLs are now thought to be distributed more widely

in the plant kingdom with more structural diversity

than had ever been previously thought

Both CCA and CRLL showed a similar extent of

homology (30–40% in amino acid identity) to other

JRLs Although the complete amino acid sequence has

not yet been determined for CRLL, a high extent of

intramolecular homology, i.e between N-terminal and

C-terminal CRDs, is also evident for both CCA and

CRLL (> 35%) [19,21] Since Greek key motif 3, a

region assumed to form a carbohydrate-binding site, is

highly conserved in both N-terminal and C-terminal

CRDs in CCA and CRLL, all of these CRDs are

likely to maintain a sugar-binding function Thus,

dis-tribution of a similar type of molecule in taxonomically

unrelated plants raises a basic question about the

bio-logical significance of tandem repeat-type mJRLs In

this context, there are some lines of evidence that

cer-tain mJRLs are induced by treatment with methyl

jasmonate or by salt stress [12,23] This suggests that

mJRLs have some defensive roles in the plant body

However, there is no clear evidence for this hypothesis

or no such report for CCA and CRLL To understand

their functions in plants, it is essential to elucidate

their biochemical properties in terms of carbohydrate-binding specificities

For this purpose, we recently developed an automa-ted frontal affinity chromatography (FAC) system, which enables us to analyse lectin–oligosaccharide interactions in a high-throughput manner [24] Signifi-cant advantages of FAC include high sensitivity and reproducibility In addition, the method is well suited for determination of dissociation constants (Kds) for relatively low-affinity binding (e.g Kd> 10)6m), thus making it practically advantageous for analysis of lectin–oligosaccharide interactions FAC was originally developed by Kasai et al [25], reinforced by Hirabaya-shi et al [26], and proved to be an effective alternative successfully applied to comparative analysis of animal lectins with a set of fluorescently labelled glycans [27] Other investigators also used an FAC system equipped with an MS detector for analysis of mushroom lectins, and demonstrated its efficiency [28,29]

In this present study we applied two tandem repeat-type mJRLs, CCA and CRLL, to this automated FAC system As a result, both conserved and divergent fea-tures of these taxonomically unrelated mJRLs became evident

Results

Evaluation of the lectin columns CCA and CRLL were purified by affinity chromatog-raphy on asialofetuin and mannose–agarose columns, respectively, as previously described [19,20] The thus purified proteins were immobilized on NHS-activated Sepharose 4FF, and the resulting resins were packed into miniature columns (inner diameter, 2 mm; length,

10 mm; bed volume, 31.4 lL) as described under Experimental procedures The amounts of immobilized CCA and CRLL were determined to be 2.0 and 1.1 mgÆmL)1 gel, respectively For evaluation of the prepared columns, it was necessary to determine the effective ligand content (Bt) based on the so-called

‘concentration-dependence analysis’ [26,27] For this purpose, however, none of the commercially available p-nitrophenyl (pNP) derivatives of simple saccharides tested, i.e Man-a, Man-b, Glc-a, Gal-a, Gal-b, GalNAc-a, GalNAc-b, Fuc-a, Galb1–4Glc-b, Galb1– 4GlcNAc-b, Galb1–3GalNAc-a, Glca1–4Glc-a or (Glca1–4)5-a, showed any significant affinity for these columns Since these lectins are known to show high affinity for the mannotriose structure, Mana1– 3(Mana1–6)Man [19,20], we tested methotrexate (MTX)-derivatized Man3GlcNAc2 (M3GN2-MTX, Fig 1A) previously synthesized successfully [30] to see

if it would be appropriate for the above

concentration-dependence analysis M3GN2-MTX showed strong

retardation when it was applied to these lectin columns

at the concentration of 5 lm, whereas it showed no

significant binding to a BSA–agarose column

(2.6 mgÆmL)1, data not shown) Hence,

concentration-dependence analysis was performed with

M3GN2-MTX at various concentrations ranging from 5 to

20 lm (Fig 1B,C) As a result, Bt and Kdvalues were

determined to be 1.49 nmol and 1.2· 10)5m,

respect-ively, for the CCA column, and 0.81 nmol and

2.0· 10)6m, respectively, for the CRLL column

Based on these data, availability of the CCA and

CRLL columns was calculated to be 39 and 40%,

respectively Specifications obtained for these columns

are summarized in Table 1

As regards the detection limit of low-affinity bind-ing, such as those for the pNP-sugars described above,

we found approximately 2 lL of experimental error in the V–V0 value in the present FAC system considering the data collection interval (1 s) and the flow rate (0.125 mLÆmin)1) Under such conditions, low-affinity saccharides having Kd values > 7.5· 10)4m and

> 4.1· 10)4m for the above CCA and CRLL col-umns, respectively, cannot be precisely characterized

On the other hand, the maximum V–V0 value measur-able in the present system is at least 120 lL, which corresponds to Kd values of 1.3· 10)5m and 6.8· 10)6m for the CCA and CRLL columns, respectively Thus, a dynamic range of 60-fold is achieved by using these columns with no change in lig-and concentrations

Fig 1 Determination of Bt values (A) Struc-tural formula of MTX-derivatized Man3Glc-NAc2 (M3GN2-MTX), which was used for concentration-dependence analysis For det-ermination of Btvalues for the immobilized CCA (B) and CRLL (C), M3GN2-MTX was diluted to various concentrations (5–20 l M ) and applied to each column The solid lines and dotted lines indicate elution profiles of M3GN2-MTX and control sugar (pNP-Lac-tose), respectively (left) Woolf–Hofstee-type plots were made by using V–V0values (right) For details, see text.

Table 1 Specifications of CCA- and CRLL-immobilized columns used in this study.

Lectin name Origin Immobilized (mg ⁄ mL) Bt (nmol) Availability (%) r 2 a M3GN2-MTX Kd( M )

a

Reliability of lines obtained as a result of Woolf–Hofstee-type plot in each concentration-dependence analysis.

Overall features of oligosaccharide specificities

of CCA and CRLL

In order to briefly profile oligosaccharide specificities

of CCA and CRLL from a global viewpoint, we

pre-pared a panel of 102 pyridylaminated (PA) glycans

including 55 N-linked glycans and 38 glycolipid-type

glycans (Fig 2) For the determination of Kd,

retarda-tion of the eluretarda-tion front relative to that of PA-lactose,

i.e V–V0, was measured for each analyte solution

dilu-ted to either 2.5 or 5.0 nm The amount of glycan

required for determination of reliable Kd value was

< 3 pmol, which is much smaller (< 10)3) compared

with other methods Since the concentrations are much

lower than Kd values assumed for the present lectin

columns as described (>
10)5m), Kd values could

be calculated according to Eqn (2) by using observed

V–V0 values in a manner independent of [A]0 For the

sake of comparison, bar graph representation was

made in terms of affinity constant (Ka) in Fig 3

Obvi-ously, both CCA and CRLL showed affinity for a

wide range of N-linked glycans, but not at all for

glycolipid-type glycans, which lack mannose This

observation is reasonable, because these lectins were

characterized as mannose-specific lectins Thus, global

features of CCA and CRLL are apparently similar,

but detailed features are different as described below

Comparison of fine specificities

High-mannose-type N-glycans

Chromatograms obtained for N-linked type glycans

are shown in Fig 4 In the case of CCA (Fig 4A), the

strongest affinity was observed for 005 (designated

M5, Kd¼ 1.0 · 10)5m), followed by 007 (M6,

1.2· 10)5m) and 003 (M3, 1.4· 10)5m) On the

other hand, CCA showed no affinity toward 001 (M2)

These results indicate that the Mana1–3Manb

struc-ture is essential for CCA binding and that the removal

of a1–3Man abolished the affinity for CCA Because

CCA could recognize 004 (M4, 2.6· 10)5m), which

lacks the depicted Mana1–3Manb structure, it proved

to have significant affinity to Mana1–3Mana, too In

contrast, removal of a1–6Man from M3 (compare 002

and 003) had rather a small effect on affinity (78%

rel-ative to 003) This observation agreed with pervious

analysis by means of haemagglutination inhibition

assay [20,22], isothermal titration caloriemetry and

enzyme-linked lectin assay (unpublished data) toward

simple saccharides The binding affinity toward CCA

was much reduced, when the nonreducing terminal

Man of the Mana1–3Manb structure was modified

by a1–2Man (008–014, corresponding to M7–9) The

tendency was confirmed by comparison of M6 isomers,

006 and 007; i.e their affinities relative to that of 005 were 36% and 83%, respectively

CRLL also showed significant affinity for relatively small high-mannose type glycans, except for 001 and

004 (Figs 3 and 4B) CRLL showed moderate affinity for an M2 saccharide, 002 (Kd¼ 4.7 · 10)5m), but not at all for its isomer, 001 Moreover, CRLL did not bind to 004, which lacks the Mana1–3Manb structure These results indicate that the core Mana1–3Manb structure forms an essential unit for CRLL recogni-tion Considering the inability of CRLL to bind to

004, the terminal Mana1–3Mana cannot substitute for the core Mana1–3Manb structure, unlike the case for CCA However, CRLL showed somewhat (30%) higher affinity for 005 (Kd¼ 3.0 · 10)5m) than for

003 (3.9· 10)5m) By comparison between 002 (M2, 4.7· 10)5m) and 003 (3.9· 10)5m), it is clear that addition of the Mana1–6Manb branch had only a small effect, if any, on CRLL–glycan interaction CRLL also showed a significant difference in affinity for M6 isomers (006 and 007) When compared with their parental molecule, M5 (005, Kd¼ 3.0 · 10)5m), the addition of a1–2Man to the Mana1–3Manb branch resulted in almost complete loss of affinity; whereas that to the Mana1–3Mana1–6Manb branch still enhanced the affinity (2.4· 10)5m) The tendency observed here is essentially the same as that observed for CCA However, the effect of the a1–2Man addition makes a clear contrast between the two lectins There-fore, the addition of a1–2Man to the core Mana1– 3Manb had rather a destructive effect on CRLL recognition

The most distinguishing feature of CRLL is its highly enhanced affinity for relatively large high-mannose-type glycans, i.e M8–9 As a matter of fact, CRLL showed the strongest affinity for 013 (M8, Kd¼ 1.1 · 10)5m) and 014 (M9, 9.5· 10)6m), whereas CCA could not bind to these large saccharides at all Binding ability of CRLL to large high-mannose-type glycans was consis-tent with the results obtained by haemagglutination inhibition assay using glycopeptides [19] Since these saccharides share two common structures, i.e Mana1– 2Mana1–2Mana1–3Manb (M2M2M3Mb) and Mana1– 2Mana1–3Mana1–6Manb (M2M3M6Mb), coincidence

of these extended structural units may contribute to the observed high affinity in CRLL In this regard, CRLL showed 1.9-times stronger affinity for an M7 saccharide,

009, containing the M2M2M3Mb unit (Kd¼ 4.1 ·

10)5m) than for its M7 isomer, 010, containing the M2M3M6Mb unit (7.9· 10)5m) Similar results were observed for M8 isomers, 012 (Kd¼ 3.0 · 10)5m) con-taining the M2M2M3Mb unit and 011 (4.9· 10)5m)

Fig 2 Schematic representation of oligosaccharide structures Note that the reducing terminal is pyridylaminated for FAC analysis Symbols used to represent pyranose rings of monosaccharides are shown in the box at the bottom of the figure Anomeric carbon, i.e position 1, is placed at the right side, and 2, 3, 4… are placed clockwise Thin and thick bars represent a and b linkage, respectively.

containing the M2M3M6Mb unit In contrast, the

increase in affinity was relatively small, when a1–2Man

was added to the M6M6Mb unit; i.e compare M8

iso-mers, 011 (4.9· 10)5m) and 012 (3.0· 10)5m) with

their parental M7 isomers, 010 (7.9· 10)5m) and 090

(4.1· 10)5m), respectively Since the increase was

approximately 1.5-fold in these cases, the addition of the terminal a1–2Man to the M6M6Mb unit still made some contribution to affinity enhancement In this context, the strongest contribution of a1–2Man was observed when it was added to the M2M3Mb unit: compare 010 with 013, and 011 with 014 (affinity

Fig 3 Bar graph representation of affinity

constants (Ka) of CCA (left) and CRLL (right)

toward N-linked glycans The small Arabic

figures in the centre correspond to sugar

numbers indicated in Fig 2; whereas large

Roman figures on the left side of graphs

represent types of glycans:

high-mannose-type (I), agalacto-high-mannose-type (II),

galactosylated-type (III) and sialylated-galactosylated-type (IV) N-linked

glycans, glycolipid-type glycans (V), and

others (VI).

Fig 4 Elution profiles of N-linked glycans obtained with CCA (A) and CRLL (B) columns Chromatograms of N-linked glycans are shown in the order of sugar numbers together with retardation volumes (upper, V–V0, lL) and dissociation constants (lower, Kd, M ) For the sake of convenience, the elution pattern of each saccharide is overlaid with that of PA-lactose, which has no affinity for either lectin, i.e the negative control.

enhanced by 7.2 and 5.2 times, respectively) In the

remaining case of the a1–2Man addition, i.e to the

M3M6Mb unit, the effect was rather intermediate

(approximately 3.5 times), when the affinity of 013 and

014 were compared with that of their parent saccha-rides, 009 and 012, respectively These results indicate that the presence of nonreducing end mannose in the M2M2M3Mb unit plays a dominant role in the strong

Fig 4 (Continued).

interaction between CRLL and large

high-mannose-type glycans, whereas M2M3M6Mb and M2M6M6Mb

contribute to lesser extents (i.e M2M3M6Mb >

M2M6M6Mb) From a practical viewpoint, glycans

having both M2M2M3Mb and M2M3M6Mb units

show the highest affinity for CRLL

Complex-type glycans

In the present study, both CCA and CRLL were found

to bind to several complex-type N-linked glycans, too

As these lectins were previously characterized as

man-nose-specific lectins [19,20], possible reasons for this

discrepancy should be given In the case of CCA, it

showed significant affinity for 101 (Kd¼ 2.2 · 10)5m),

201 (2.8· 10)5m), 301 (1.6· 10)5m), and 401

(2.3· 10)5m); whereas it showed lower affinity for

their position isomers, i.e 102 (3.5· 10)5m), 302

(3.7· 10)5m), and 402 (5.5 · 10)5m) relative to 101,

301 and 401, respectively This observation indicates

that CCA prefers mono-antennary, complex-type

N-linked glycans, which have a1–6 branch, or

nonsub-stituted a1–3 Man Such a feature is consistent with

the idea that Mana1–3Manb forms the core

recogni-tion unit On the other hand, we also found that CCA

showed significant affinity for bi-antennary glycans,

i.e 103 (Kd¼ 2.9 · 10)5m), 202 (5.2· 10)5m), 304

(3.4· 10)5m), 307 (3.7 · 10)5m), 403 (5.0· 10)5m),

404 (5.4· 10)5m), and 405 (5.3· 10)5m) Their

affinities for CCA were similar (3–5· 10)5m), but

somewhat (40–80%) reduced in comparison with those

for the respective parental mono-antennary glycans,

i.e 101, 201, 301, and 401 These results indicate that

modification of a1–3Man is never detrimental This

may explain why CCA could bind to

asialofetuin-agarose [20], because this glycoprotein contains

bi-antennary, complex-type glycans [31] On the other

hand, tri-antennary glycans showed no affinity for

CCA Therefore, C4-OH group of a1–3Man is

essen-tial for CCA recognition

Similar to CCA, CRLL also showed significant

binding to mono-antennary, complex-type N-linked

glycans having the a1–6 branch, i.e 101 (Kd¼

3.9· 10)5m), 201 (2.7· 10)5m), 301 (4.1· 10)5m),

and 401 (2.4· 10)5m) Unlike CCA, however, CRLL

did not show affinity at all for their position isomers

(102, 302, and 402) Moreover, CRLL had no affinity

for bi-antennary glycans (e.g 103, 104, 202, 203, 303,

304, 307, 308, 403, 404, 405, and 406) Neither

tri-antennary nor tetra-tri-antennary N-linked glycans were

targets for CRLL, either Therefore, CRLL is stricter

than CCA in that the former never permits

substitu-tion of a1–3Man in complex-type glycans

In the present study, the influence of a1–6 (core) fucosylation on lectin–glycan interaction could also be examined by comparison between 003 and 015, 101 and 201, 103 and 202, 104 and 203, 301 and 401, 302 and 402, 304 and 403, and 307 and 405 (Figs 3 and 4)

In the case of CCA, a1–6 (core) fucosylation slightly reduced the affinity; for example, compare 101 (Kd¼ 2.2· 10)5m) with 201 (2.8 · 10)5m) With CRLL, however, this type of fucosylation enhanced the affinity

by 1.2–1.7 times; compare 101 (Kd¼ 3.9 · 10)5m) with 201 (2.7· 10)5m) The effect of a1–6 fucosyla-tion was significant, but not very drastic in both cases

So, the presence of a1–6Fuc does not have any essen-tial role for the recognition of these mJRLs

Oligosaccharides 501–505 represent sialylated gly-cans Among them, bi-antennary glycans (501, 502, and 503) were recognized by CCA to some degree, whereas none of them showed significant affinity for CRLL In the case of CCA, it is clear that the effect

of sialylation of bi-antennary glycans was different between mono-sialylated isomers i.e 501 (Kd¼ 1.1· 10)4m) and 502 (4.5· 10)5m) By comparison with nonsialylated glycan, 307 (3.7· 10)5m), the inhi-bitory effect of sialylation was more drastic on the a1–3 branch (affinity reduced to 34%) than on the a1–6 branch (to 82%) Again, this result is consistent with the above observation that Mana1–3Manb is an essential unit for CCA recognition

Summary of FAC analysis of CCA and CRLL The sugar-binding properties of CCA described above may be summarized as follows: (a) CCA shows affinity for mannose-containing N-linked glycans, but not for glycolipid-type glycans; (b) it binds to relatively small high-mannose-type glycans, which contain a nonsubsti-tuted a1–3Man residue in the tri-mannosyl core struc-ture; (c) the binding is greatly diminished by the addition of a1–2Man residue(s) to the a1–3Man; (d) CCA can also bind to mono-, bi-antennary N-liked glycans with varied affinities, but not to tri- and tetra-antennary ones

On the other hand, CRLL has the following fea-tures: (a) similar to CCA, CRLL shows affinity only for N-linked glycans but not for glycolipid-type ones; (b) unlike CCA, however, CRLL binds to relatively large high-mannose-type glycans with much increased affinity; (c) a1–2Man extension in the M2M2M3Mb unit makes the strongest contribution to such high affinity for the large high-mannose type glycans; (d) affinity enhancement is also supported by a1–2Man extension in the other units M2M3M6Mb and M2M6M6Mb; (e) CRLL can bind only to a1–6

branched mono-antennary, complex-type N-linked

gly-cans, but never to a1–3 branched mono-, bi-, tri- and

tetra-antennary glycans

Discussion

By using our recently developed FAC system, we

ana-lysed detailed sugar-binding specificities of CCA and

CRLL The advantage of FAC in sensitivity, economy

and reliability enabled us to reveal fine specificity of

these lectins Based on the results, both conserved and

divergent properties were reveled for these two

mJRLs For relatively small high-mannose-type

gly-cans, CCA and CRLL showed similar sugar-binding

profiles: they bound to Mana1–3Manb as a main

recognition unit, and the affinity was reduced by the

addition of an a1–2Man residue to the Mana1–3Manb

structure However, according to the X-ray

crystallo-graphic study on Heltuba, another mJRL, in complex

with a1–3 mannobiose, no direct hydrogen bond was found at the O2 atom of mannose [5] Thus, substi-tution of 2-OH with a1–2Man seems to have no destructive effect on interactions On the other hand,

a modelling study on Artocarpin, another mJRL, with ManNAc also revealed that severe steric hindrance occurs between the N-acetyl group of ManNAc and the residues in the loops b1-b2 and b11-b12, which construct the primary-binding site [32] At a glance, the amino acid sequences around the region critical for sugar-binding function are tightly conserved among mJRLs (Fig 5) Therefore, the reason why the affinity was diminished by the addition of a1–2Man residue(s) to the core Mana1–3Manb unit can prob-ably be attributed to significant steric hindrance by the residue

As regards the relatively large high-mannose-type glycans (M7–M9), which showed much enhanced affin-ity only for CRLL, considerable steric hindrance will

Fig 5 Sequence alignment of mJRLs Individual CRDs of CCA (N- and C-domains) and partial amino acid sequences reported for CRLL [19] are aligned with other representative mJRLs Residues conserved in all mJRLs are indicated in bold letters Secondary structure elements (b-sheet) and a1–3 mannobiose-binding residues reported for Heltuba are indicated by arrows and filled circles, respectively [5].