Carbohydrate specificity of lectin , purified from the fruiting bodies of Mycena pura / Fr . / Kumm . and its use in histochemical investigation

Aim. The purpose of this investigation was to research carbohydrate specificity of a new lectin from fruiting body of Mycena pura and possibilities of its application in histochemical studies. Methods. The lectin has been purified by affinity chromatography on «îvomucine». The lectin carbohydrate specificity has been determined by a reaction of inhibiting haemagglutination by haptens. Histological materials were fixed in 4 % neutral formalin solution. Alkaline phosphatase was revealed in the cryostat unfixed microscopical sections. Results. The lectin yield from fresh fruit bodies of raw material was 9 mg/kg. Mol. mass of the lectin is 40 kDa. The lectin poorly interacted with D-glucose and D-mannose in contrast to lectins from Pisum sativum and Leucojum vernum. The peculiarity of this lectin is its strong interaction with alkaline phosphatase, the highest among twenty tested lectins. However, the receptors for Mycena lectin binding in mammalian tissues are not limited by this enzyme being presented also by glycoconjugates of another structure, as it was shown for fetus calf small intestine and kidney of rat. Conclusions. An important role in the lectin interaction with glycoproteins probably belongs to the disaccharide links of GlcNAcb(1-2)Mana(1-6) or GlcNAcb(12)Mana(1-2), which not necessarily are terminal.

In tro duc tion.Lectins are a group of pro teins of non-im mune or i gin, no ta ble for fea tures of re vers ible and se lec tive bind ing to car bo hy drates and car bo hydrate de ter mi nants of biopolymers, with out chang ing their co va lent struc ture [1].Most liv ing or gan isms have been found to con tain lectins, per form ing var i ous func -tions, based on the pro cesses of rec og niz ing car bo hydrate struc tures in macromolecules.The end of the 1980's wit nessed the dis cov ery of the fact that the in tegral com plex of dif fer ent lectins func tions in both vege ta ble and an i mal or gan isms.Act ing as com pan ions, lectins reg u late the intracellular glycoprotein movement.For in stance, calnexin (mem brane-bound lectin of endoplasmatic re tic u lum) func tions si mul ta neously with its sol u ble an a logue calreticulin as a part of the sys tem of reg u lat ing glycoprotein qual ity.Galectins (galactose-spe cific lectins of an i mal or i gin) act as modu la tors of in ter ac tions be tween the sub strate and the cell, and are re quired for nor mal func tion ing of the programme of dif fer en ti a tion and growth of all multicellular an i mal or gan isms.They are ca pa ble of stim u lat ing cell pro lif er a tion and apoptosis, and par tici pate in organomorphogenesis, me tas ta sis of tu mour cells, im mune re sponse and in flam ma tory pro cesses as well as in rec og ni tion of extracellular ma trix [2].The role of lectins in mycorrhize formation between the fungal hyphae and coniferous trees was studied for true fungi, Basidiomycetes, in particular [3].
To date lectins have been widely used in his tochem is try in ves ti ga tions on the de ter mi na tion of car bohy drate de ter mi nants on the cell sur face.The lectins (la belled by peroxidase, col loi dal gold, fluorochrome), ob tained from dif fer ent liv ing or gan isms, are used for the pur poses of the above men tioned in ves ti ga tions.The study on phylo gen eti cally re mote or gan isms enhances the prob a bil ity of de ter min ing lectins of rare car bo hy drate spec i fic ity which would open new op portu ni ties for his to chem is try in ves ti ga tions.There fore, the search for, ob tain ing and anal y sis of carbohydrate specificity of new lectins is an important task.
While study ing the fruit bod ies of basidium fungi of Mycena pura /Fr./Kumm) we have dis cov ered a lectin which has not been de scribed in lit er a ture yet, and called it Mycena pura fun gus ag glu ti nin (short name -MPFA).
The aim of the cur rent work is to char ac ter ize the car bo hy drate spec i fic ity of the lectin and to an a lyze the pos si bil i ties of its use in histochemical research.
Ma te ri als and Meth ods.The fruit bod ies of the Li lac Bell Cap for the pur pose of ob tain ing lectins were col lected in Skole dis trict of the Lviv re gion.They were trans ported to the lab o ra tory on the same day as col lec tion.
The iso la tion of lectins from the fruit bod ies of the Li lac Bell Cap was per formed us ing the af fin ity chroma tog ra phy on ovomucin as the sorbent us ing a pre viously de scribed method [4].The scheme of lectin pu rifi ca tion in cluded the ex trac tion of shred ded fruit bod ies us ing the buf fered phys i o log i cal so lu tion (BPS), with sub se quent acid i fi ca tion to pH 4.5, and re duc tion of the ex tract to pH 8.4 with the re moval of pre cip i tates formed, pre cip i ta tion of pro teins with am mo nium sulphate (600 g/l), af fin ity chro ma tog ra phy on ovomucin, ad di tional pu ri fi ca tion on DEAE-Toyopearl in 0.1 M phos phate buffer so lu tion, pH 7.0, concentration and and freeze-drying of the purified preparation.
The prep a ra tion pu rity was es ti mated us ing disk-elec tro pho re sis in 10% polyacrylamide gel (PAAG) in al ka line buffer sys tem (pH 8.6).Af ter the elec tro pho re sis in two par al lel tubes, the gel in one of them was stained with coomassie R-250, the gel from the other was cut in 5 mm pieces and ho mog e nized with 0.25 BPS, pH 7.4 with sub se quent de ter mi na tion of hemagglutination ti tre in the extracts obtained.
The car bo hy drate spec i fic ity of lectins was de termined us ing the re ac tion of hemagglutination in hi bition with car bo hy drates and glycoproteins.The stepwise di lu tion of the car bo hy drate was used to de termine its min i mal con cen tra tion, which in hib its the activ ity of lectin with 1:4 titre completely [6].
The in ter ac tion with glycoproteins and poly sac charides was de fined us ing the gly co gen of por cine liver, ovomucoid, thrice re-crys tal lized ovalbumin (Biolar, Lat via), and yeast mannan [7].Group-spe cific substances H, A, and B were iso lated from the cyst liq uid, ob tained af ter the sur gery on the ova ries of pa tients' who have cor re spond ing blood groups.The men tioned sub stances were pu ri fied by the method, de scribed in [1].The pu ri fied transferrin and orozomucoid were kindly pro vided by Professor M.D. Lutsyk.
The con ju gates of lectins with horse rad ish peroxidase were pre pared by the method, de scribed in [8].
Al ka line phosphatase ac tiv ity was de ter mined in cryostat-un fixed mi cro scopic sec tions us ing a method orig i nally in tended for cytochemical re search [9] and adapted by us for the small in tes ti nal tis sue of 6-month-old calf fe tuses and rat kid ney tis sue.The incu ba tion so lu tion con tained 10 mg naphtol-AS-MX phos phate (Sigma, USA), 10 mg fast blue PP (Fluka, Ger many) in 10 ml 0.08 M tris-HCl buffer so lu tion, pH 9.2, the in cu ba tion was per formed us ing the mi croscope con trol to ob serve the ap pear ance of blue granules (5-10 min), then the sec tions were washed with the dis tilled wa ter and put into gel a tin-glyc erol.At the same time the sec tions were stained with the con ju gate of horse rad ish peroxidase-lectin of Mycena pura /Fr./Kumm.
The histological ma te ri als were fix ated in 4% neutral for ma lin in or der to in ves ti gate the in ter ac tion of lectins, stained with peroxidase, and the prep a ra tions of rat co lon.The 5-7 µm sec tions were stained with hematoxylin and eosin for gen eral morphology [10].
The mi cros copy and pho tog ra phy of the prep a ra tions were per formed us ing the photomicroscope MBI 15-2.
Re sults and Dis cus sion.From 1 kg of Mycena pura fresh fruit bod ies, 9 mg of the pu ri fied lectin was ob tained.This is about 59% of the the o ret i cal out put based on cal cu la tion of the ac tiv ity in an ini tial extract.
The in ten sity of stained stripes and the data of the ag glu ti na tion (per formed in the par al lel gel tube) during elec tro pho re sis in 10% PAAG in an al ka line buffer sys tem (pH 8.6) al low the con clu sion that the pu ri fied prep a ra tion con tains about 90% lectin (Fig. 1).This amount is suf fi cient to per form the ma jor ity of histochemical investigations.
An area of M r » 40kDa was ob served dur ing electro pho re sis in 15% PAAG in the pres ence of 0.1 DS-Na pu ri fied lectin of M. pura (Fig. 2).
The ag glu ti na tion ca pac ity of the pu ri fied Mycena lectin rel a tive to eryth ro cytes of hu mans and an i mals is pre sented in Ta ble 1.The spec i fic ity of Mycena lectin is its ca pac ity of hemolyzing the eryth ro cytes of rabbits, hu mans, and dogs in the con cen tra tion of 1 mg/ml along with ag glu ti na tion, there fore, it may be con sidered to be a bifunctional lectin.The hemolysis (not agglu ti na tion) is in hib ited in the pres ence of poly eth yl ene gly col with M r = 3000 Da, which en ables de ter min ing both the hemagglutination titre and carbohydrate specificity of the lectin.
The in ves ti ga tion of the in ter ac tion of the lectin (MPFA) with car bo hy drates re vealed that it has rather poor in ter ac tion with D-glu cose and D-mannose.There fore, we de cided to com pare its car bo hy drate spec i fic ity with that of the lectin of pea (Pisum sativum ag glu ti nin, PSA, Fabaceae fam ily), and the lectin of a snow flake (Leucojus vernus ag glu ti nin, LVA, Amaryllidaceae fam ily), which are the rep re sen ta tives of two dif fer ent sub groups of D-mannose-specific lectins [11].The three in ves ti gated lectins (PSA, LVA and MPFA) have better bind ing to com pound oligosaccharide struc tures, which are glycoconjugate com po nents.In stead, their in ter ac tion with some substances is con sid er ably dif fer ent.For in stance, the yeast mannan in ter ac tion with LVA is very strong, PSA -me dium, and the Mycena lectin -com pletely ab sent.PSA binds strongly to bo vine thy ro glob u lin and sheep submaxillary mucin, while the in ter ac tion of LVA and MPFA with the lat ter is me dium.The Mycena lectin strongly in ter acts with calf in tes ti nal al ka line phosphatase, while PSA has poor in ter ac tion with it, and LVA does not in ter act at all.The abovementioned fact tes ti fies to higher complementarity of the cen tres of car bo hy drate in ter ac tion of the Mycena lectin, PSA and LVA to wards dif fer ent car bo hy drates of complex and branched structure (Table 3).
There are lit er a ture data, prov ing the pres ence of alka line phosphatase (E.C.3.1.3.1) in all the tis sues and liq uids of the or gan ism, ex cept for the ves sel walls and hyaline car ti lage.This en zyme is abun dant in the ep ithe lium of the small in tes tine wall, flexu ous re nal tubules, bone tis sue, breast glands dur ing lac ta tion, and in leu co cytes [12].Al ka line phosphatase of the calf small in tes tine is zinc-con tain ing two-chain The interaction of Mycena lectin with some mono-and disaccharides glycoprotein with M r = 65 kDa, that has two sites of glycosylation -near asparagine-249 con tain ing a ba sic glycosylres i due is with two an ten nas and con tains D-galactose, D-glucosamine, and D-mannose, while glycosylated res i due near asparagine-410 is with four an ten nas and con tains L-fucose (Fig. 3).To tal amount of car bo hy drates in the phosphatase is from 8 to 17% (de pend ing on the type and lo cal iza tion of the en zyme) [13].The struc tures of the men tioned en zyme in humans and mam mals have some dif fer ences, but they are not con sid er able.Due to a high amount and va ri ety of car bo hy drates in the phosphatase one may ex pect its inter ac tion with many lectins, among which there are some with different carbohydrate specificity.Tak ing the above men tioned into con sid er ation, we have stud ied the in ter ac tion of a pu ri fied en zyme with the lectins, which are used in histochemical in ves ti gations the most fre quently and the lectins, which were first ob tained in our lab o ra tory.The re sults, pre sented in Ta ble 4, dem on strate that many lectins re ally bind to the phosphatase, in par tic u lar, there were strong in terac tions with the lectins of soy bean and ed ible snail, while the al ka line phosphatase was the stron gest in hibi tor of the ac tiv ity for Mycena pura lectin.The lectins of Amaryllidaceae fam ily (snow drop and snow flake) and, un ex pect edly, the lectin of mis tle toe do not interact with the alkaline phosphatase.
The re ac tion for al ka line phosphatase per formed as a con trol in non-fixed frozen sec tions of small in tes tine of 6-month-old calf foe tus dem on strated the pres ence of the en zyme only in enterocytes with the ep i the lial plate bor der of villi and crypts, while the re ac tion with the stained Mycena lectin on the sec tions re vealed a sim i lar pat tern of the in ter ac tion with slight dif ferences.The re ac tion for al ka line phosphatase was more in tense in enterocytes of both villi and crypts, while only some enterocytes of crypts re acted to Mycena lectin (Fig. 4, a, b).An other ver i fi ca tion of the as sump tion re gard ing the se lec tiv ity of Mycena lectin bind ing to car bo hydrate de ter mi nants, pres ent in the mol e cule of al ka line phosphatase, was per formed in the tis sues of rat kidney, which is rich in var i ous car bo hy drate re cep tors.In this ex per i ment we iden ti fied the ac tiv ity of al ka line phosphatase in the com po si tion of brush bor der of prox i mal re nal tu bules of new-born rats (Fig. 5), whereas the re cep tors of Mycena lectin were local ised in the cy to plasm of dif fer ent pop u la tions of nephrocytes with the ten dency to ac cu mu lat ing in the api cal part and on the luminal sur face of the even tual am poules cells, S-like bod ies, and the ba sic parts of the collapsible kidney channels.
The data ob tained may tes tify to the fact that the action of sub strates for bind ing MPFA lectin is much wider and is not re stricted only by the ac tiv ity of al kaline phosphatase.They may be pre sented in kid neys by other glycoconjugates of com plex struc ture with ter minal de ter mi nants DMan/DGlc and DGlcNAc, in cluding glycogen.
To de tect these glycoconjugates, lectin bind ing with car bo hy drate re cep tors was ex am ined us ing histological prep a ra tions with a row of con trol re actions on mi cro scopic sec tions of rat small in tes tine using car bo hy drate and glycoprotein inhibitors.
Our as sump tion on neg a tive re sults of the con trol re ac tion for the de ter mi na tion of prob a ble en dog e nous peroxidase was proven.
The re ac tion with the stained Mycena lectin was clearly pos i tive.The pres ence of 5% so lu tion of a-methyl-D-galactopyranoside, with which the lectin does not in ter act, did not re sult in the in hi bi tion of the re ac tion.The pres ence of 5% so lu tion of a-methyl-D-mannopyranoside (»250 mM so lu tion), with which the lectin in ter acts, re sulted in weaker re action, but there was no com plete in hi bi tion of the re action.It proves that the abovementioned car bo hy drate is a poor in hib i tor of Mycena lectin.The pres ence of 1% ovalbumin (»0.23 millimolar so lu tion or in mo lar concen tra tion, which is 250:0.23 = 1087-fold less for that of a-methyl-D-manopyranoside) weak ened the re action, but its level was ap prox i mately equal to that of the pres ence of 5% so lu tion of a-methyl-D-manopyranoside.And only in the presence of 1% so lu tion of al ka line phosphatase of calf small in tes tine the re ac tion was blocked com pletely.The con cen tra tion of the lat ter is 0.15 mM which is close to the con cen tra tion of ovalbumin, but it is approx i mately 1623 times lower than the mo lar con centra tion of a-methyl-D-mannopyranoside.One must take into ac count that, on av er age, only one-sixth of the al ka line phosphatase mol e cule is a car bo hy drate fragment.In other words, the oligosaccharide chain, isolated in a pure form, may be al most a ten thou sand times (1623•6 = 9740) stron ger in hib i tor than a-methyl-D-mannopyranoside.
Egg al bu min, which has a me dium MPFA in hib i tory ac tiv ity, con sists of 3.2% car bo hy drate that forms three and four antennate glycan links; there can be at least 6 dif fer ent vari ants, 2 of which are pre sented in Fig. 6 [14].
The com par i son of struc tures of glycan res i dues of al ka line phosphatase and ovalbumin dem on strates that the in ner part of oligosaccharide chains (disaccharide links GlcNAcb(1-2)Mana(1-6) or GlcNAcb(1-2)Mana(1-2)) is sim i lar for them, while ter mi nal car bo hy drates are dif fer ent.As for al ka line phosphatase, the res i dues of disaccharides Gala(1-3)Galb(1-4), which do not in hibit the in ter action of the lectin and carbohydrate, are ter mi nal.It proves that the ac tive cen tre of Mycena lectin is comple men tary to com plex oligosaccharide struc tures, prob a bly, branched oli go sac cha rides of four-antennate type, where disaccharide links GlcNAcb(1-2)Mana(1-6) play a sig nif i cant role in the lectin-carbohydrate interaction.
Be cause of the pres ence of al ka line phosphatase in enterocytes of the large and small in tes tine and in ter action with this en zyme of the ma jor ity of tested lectins, it is much more in for ma tive for study of histological prep a ra tions of the co lon is the use of lectins that do not in ter act with al ka line phosphatase.
The lectins of snow flake and snow drop al most do not in ter act with histological sec tions of rat co lon.The "multimannose type" oli go sac cha rides with ter mi nal Mana(1-3)Man-res i dues are known for the high est inhi bi tion ac tiv ity for the lectin of the snow drop.This lectin also ac tively in ter acts with oli go sac cha rides, con tain ing branched a(1-6)-bound res i dues of glu cose or mannose, but it does not in ter act with branched res idues, con tain ing Gal, GalNAc, and Fuc.The lectin of the snow flake is char ac ter ized by sim i lar spec i fic ity.There fore, one can firmly state that there are no such carbohydrate structures in the colon.
Other lectins, used for stain ing histological structures of the co lon, may in ter act with both al ka line phosphatase and other car bo hy drate re cep tors, thus, their re sponse is ambiguous.
1.The in ves ti ga tion of the in ter ac tion of the new lectin, pu ri fied by us from the fruit bod ies of Mycena pura, with car bo hy drates and glycoproteins re vealed that ac cord ing to the wide-spread clas si fi ca tion of lectins, this lectin may be re ferred to glucoso-(mannoso-)-spe cific ones.How ever, its fine car bo hy drate spec i fic ity dis tin guishes it from pre viously known lectins of le gumes and Amaryllidaceae fam ily (the lectins of Pisum sativum and Leucojum vernum).The re sults of in ves ti ga tion on the bind ing of the men tioned lectin to glycoproteins al low the assump tion that the im por tant part be longs to disaccharide links GlcNAcb(1-2)Mana (1)(2)(3)(4)(5)(6) or GlcNAcb(1-2)Mana(1-2), while ter mi nal res i dues of disaccharides Gala(1-3) Galb(1-4) do not abol ish the in ter ac tion of the lectin and car bo hy drates and, prob ably, are not sig nif i cant for this interaction.
2. The dis tinc tive fea ture of Mycena lectin is its strong in ter ac tion with al ka line phosphatase -the rel ative in hi bi tion of this in ter ac tion was found to be the high est among twenty in ves ti gated lectins.How ever, the sen si tiv ity of re cep tors for bind ing MPFA lectin in the tis sues of mam mals is not lim ited to al ka line phosphatase only, it may be re lated to other glycoconjugates of com plex struc ture, as it was shown on the ex am ple of rat kid ney and small intestine of calf foetus.

Fig. 4 .
Fig.4.Reaction to alkaline phosphatase (a) and staining with Mycena lectin (b) on the preparation of small intestine of 6-month-old calf foetus (x120); KBvilli cells; KK crypt goblet cells Arrows indicate: a -places of the most intense reaction to alkaline phosphatase; bsites of binding of Mycena lectin

Table 1
Mycena lectin, capable of causing the agglutination of erythrocytes, µg/ml Minimal concentration of purified Mycena lectin which causes the agglutination of erythrocytes