Structures of the neutral oligosaccharides isolated from A-active human gastric mucin.

Alkaline borohydride reductive cleavage of the mucin, purified from gastric aspirates of the secretors with blood group A, resulted in a heterogeneous population of neutral (79.7%) and acidic (20.3%) oligosaccharide alditols. Nine oligosaccharides (I-IX), ranging from 6 to 15 sugar units, have been purified from the neutral oligosaccharide fraction. Based on the results of immunological assays, sugar composition, degradation with specific exoglycosidases, and methylation analyses, we propose the following structures for these oligosaccharides: (sequence in text)

to 15 sugar units, have been purified from the neutral oligosaccharide fraction. Based on the results of immunological assays, sugar composition, degradation with specific exoglycosidases, and methylation analyses, we propose the following structures for these oligosaccharides: tionai Institute on Alcohol Abuse and Alcoholism and Grant AM-epithelium is a heterogeneous mixture of the molecules which 14743 active secretion or by passive transudation. This viscous layer consists of proteins, glycoproteins, and lipids imbibed with water and electrolytes (1)(2)(3)(4)(5). The integrity of mucus gel, essential for its physiological role as a lubricative and protective layer, depends primarily on its mucin component which is secreted by mucous cells of gastric epithelium (1,3,(6)(7)(8)(9). The mucin of human and pig stomach is a large (2 X lo6), highly glycosylated polymer composed of four subunits of equal size joined together by disulfide bridges at their nonglycosylated regions. These naked nonglycosylated protein regions are susceptible to proteolysis, and under physiological conditions, the mucin is broken down to glycoprotein subunits (5 x lo5) by pepsin (3,10,11). The glycosylated region of each glycoprotein subunit contains numerous carbohydrate side chains 0-glycosidically linked to the hydroxyl groups of seryl and threonyl residues of the protein core (1,3,12). The size and structure of the carbohydrate chains in mucins from different regions of the gastrointestinal tract varies in the same species as well as between species (13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Furthermore, considerable heterogeneity exists in the structure of carbohydrate chains of mucin within the same tissue (15,(18)(19)(20)(21)(22)(23).
As part of the project directed towards establishing the role of carbohydrate chains of mucin in gastric mucosal protection (9,24), we have begun a study on the structure of carbohydrate moiety in human gastric mucin. In a previous report, we have described the structure of neutral oligosaccharides of H-active mucin (18). Here, we present the structural characterization of nine oligosaccharides derived from A-active gastric mucin. These oligosaccharides range in size from 6 to 15 sugar units in length and represent 78% of the neutral carbohydrate chains of this mucin.

DISCUSSION
A common characteristic of mucin-type glycoproteins from various organs and species is the heterogeneous nature of their carbohydrate chains (16, 18-22, 43, 44). These chains vary in length from a single N-acetylgalactosamine residue to elongated complex structures containing as many as 20 sugar units (13-22, 31, 44-46). Although the physiological importance of variations in carbohydrate chains of mucins is far from being well understood, it is generally accepted that this feature allows for each mucin an infinite number of distinct biological functions. To determine how these variations affect the functional properties of mucins, it is necessary to establish the structures of individual carbohydrate chains.
Recently, we have shown that H-active human gastric mucin contains a t least 10 different neutral carbohydrate chains (18). The data presented here indicate that a high degree of structural heterogeneity also exists in the carbohydrate chains of A-active human gastric mucin. We have found that the carbohydrate chains released from this mucin by alkaline borohydride treatment consist of neutral (79.7%) and acidic (20.3%) oligosaccharides. Nine oligosaccharides, representing 62% of the total carbohydrate chain of mucin, were purified from the neutral oligosaccharide fraction. These compounds accounted for 78.2% of the neutral oligosaccharide fraction and ranged from 6 to 15 sugar units in length. The results of hemagglutination inhibition assays indicated that all oligosaccharides bore the A antigenic determinant. Furthermore, oligosaccharides 11, 111, IV, V, and VI exhibited H antigenic activity, while oligosaccharides I, V, and IX had the ability to inhibit hemagglutination in the I anti-I system. The presence in each oligosaccharide of carbohydrate sequences conforming its antigenic properties was confirmed with the aid of specific glycosidases and permethylation analysis. The data revealed that two of the isolated oligosaccharides (oligosaccharides IV and VIII) contained the unbranched core with N-acetylgalactosaminitol substituted a t C-3, while in the remaining compounds, this sugar residue was C-3,6 disubstituted. The C-6 substituent consisted of GalpldGlcNAc disaccharide in oligosaccharides, I, V, and IX, Fuc~~1+2Gal~l-+3/4G1cNAc trisaccharide in oligosaccharide 11, and galactose residue in oligosaccharides 111, VI, and VII. In all oligosaccharides, C-3 on N-acetylgalactosaminitol was substituted with galactose. The results of enzymatic degradation suggested that this galactose residue served as a branching point for chains bearing A and H determinants in oligosaccharides V and VI.

The side chains bearing A determinant in the oligosaccharides I and I1 and A and H determinants in the oligosaccharides 111
and IV were shown to be p(1+3/6) linked to the Galp1-+ 3/4GlcNAc~l+3Gal@l+3GalNAc-ol tetrasaccharide. Based on these data, the following structures are proposed for the isolated oligosaccharides (Structures I-IX).
The results presented in this report, together with our previous findings (18), indicate that the neutral carbohydrate chains which predominate in human gastric mucin constitute a spectrum of oligosaccharides composed from 4 to 15 sugar units arranged in linear or branched fashion. The majority of the chains are highly branched and contain up to three antennae. The antennae bearing A or H antigenic determinants contain two types of carbohydrate chains, type 1 (GalP1 4GlcNAc) and type 2 (Galpl44GlcNAc), with the former being predominant. The predominance of type 1 carbohydrate chains in mucin-type glycoproteins in man has been reported previously (15,16), and type 2 chains were found in mucins of pig, horse, dog, and sheep stomach (16,17,20,22). The core portion of the neutral carbohydrate chains of human gastric mucin appears to consist of either Galpl-3GalNAc disaccharide or Ga1~1-+3/4GlcNAc~l-+3Gal@l-+3GalNAc tetrasaccharide. In simple oligosaccharides from H-active mucin, the distal galactose of this core is substituted at C-2 by n-L-fucose and in A-active oligosaccharides by GalNAcal-+ 3(Fuc~t1+2). In more complex structures, the core distal galactose gives rise to additional branches by p( 1-3/61 substitution with N-acetylglucosamine. As in oligosaccharides isolated from gastric mucins of other species (16,17,20, 221, further branching in carbohydrate chains of human gastric mucin occurs C-6 of the N-acetylgalactosamine residue involved in the 0-glycosyl linkage to serine/threonine of the protein core. Our data indicate that in the neutral carbohydrate chains, this C-6 substituent may be a single residue of galactose or N-acetylglucosamine, or it may consist of blood group H or I antigenic determinant. Since up to 30% of the carbohydrate chains in human gastric mucin are represented by the sulfated and/or sialylated chains, it remains to be determined whether these chains possess the structures homologous to neutral oligosaccharides described here and elsewhere (18). Only then will we be able to fully ascertain how the structural heterogeneity in the carbohydrate chains of gastric mucin is translated into its biological functions to which this mucin is destined.

GalNAc~lt3lfucolt2lGalgl-3/4R
A-antigemc determinant.    The A a c t l v i t y was masured agalnrt 8 units a f a n t i -A serum, H a c t i v i t y d g a i n s t 2 u n i t s of a n t ? -H l e c t i n , and I a c t l v i t y a g a l n r t 4 u n i t s o f " B a j " a n t l -l serum.  The 011goIacchwide I 1 e x h i b i t e d A and H a n t l g e n i c a c t i w t y , and was s u s c e p t i b l e t o the dctlon of a-L-fUCOSidale and a-N-acetylgalactosaminidare (Table IY). A f t e r z -t -f u wo f galactose and one residue Of N-acetylglucosamine (Table  IV,  and N-acetylgalactoramine. C-2, C-3, C-2.3 and C-3,6 r u b r t l t u t e d g a l a c t a r e . C-314 substituted N-acetylglucolmine. and C-3.6 substituted N-acetylgalactosaminrtol. P e m t h y l a t i a n a o a l y r l l o f t h e p a r t i a l l y degraded oligoraccharlde 11 (Table V I . Flg. 4U) Showed the presence of me C-3.6 and three C-3 substituted residue6 O f qdlaCtOPe. two unlubstituted residues of N-1Cetyl4dllCtOldmine. C-3 Substituted N-aCetYI-galdCtosdmlnitol. and three residues of C-3j4 subriiiuted N-acetylglucosamine,

Molar FatlOS Of the alditol acetates found ~n t h e h y d r o l y s a t e s o f p e m t h y l a t e d i n t a c t oligosaccharlder
The abovd data indicate that oligosaccharide I 1 cOntaln6 t n a n t e n n a r y s t r u c t u r e .  G a l h l f 3 1 4 G l c N A c s 1~3~I~I~3 G a l N A c -o l tetraraccharide.
The vewlts of sequentla1 degradation of OligoPaccharidez 111 and I Y are presented I"

Experlmnt
Carbohydrates     Conpanson O f p e m t h y l a t l o n d a t a from t h e p d r t l a l l y degraded OllgOLaCChaPlde I X (Table V I ) w?th that Of t h e i n t a c t compound (Table Y)