The biosynthesis of intestinal mucoproteins

These investigations have been concerned with the biosynthesis of a well-defined mucin fraction obtained by papain digestion from sheep colonic mucosal scrapings. This mucin fraction which accounted for 12.2% of the total dry weight of the tissue contained 3 anionic components. The major constituent was an electrophoretically slow-moving component (fraction H), a minor mucoprotein constituent (fraction L) was also present, and nucleic acid the fastest electrophoretically moving constituent. Fraction H, separated from other components by preparative ultracentrifugation is known to have a structure similar to that of other epithelial mucins. This is considered to consist of a central protein core (rich in serine and threonine, together 32% of the total amino acids) carrying carbohydrate side-branches (Kent and Marsden, 1963). In the carbohydrate side branches fucose (3.8%) and sialic acids (N-glycollyl- and N-acetylneuraminic acids 8.5 and 5.2% respectively) are located as terminal residues. These are attached to galactose (13.0%) and in turn to amino sugar residues (N-Hacetylglucosamine and N-acetyl- galactosamine 18.1% and 7.9% respectively as the free hexosamines). Ester sulphate (3.5%) is mainly attached to the hexosamine residues. Fraction L is apparently also a mucoprotein but different from H in that it is rich in fucose and ester sulphate and contains no sialic acid residues.
Scrapings of sheep colonic tissue incubated at 37° in an atmosphere of O2 in modified Krebs III medium (D-glucose omitted but with L-glutamine added) are metabolically active (Qo2 - 3.75). When incubated for 2&half; hr. under these conditions with radioactive substrates, the tissue readily incorporates radioactivity into the carbohydrate and peptide moieties of fraction H (fraction L and nucleic acid remaining unlabelled).
[U-14C] -D-glucose and [2-14C] -D-glucose are incorporated into mucin, 16.7% and 22.9% respectively of the total counts added being incorporated. Analysis of a typical sample of mucin isolated from incubation with [2-14C] -D-glucose (specific activity of mucin formed, 11.950 counts/min./mg.) gave the following distribution of counts: N-acetylneuraminic acid 8.34%, N-glycollylneuraminic acid O.95%, fucose 10.2%, galactose 14.5%, glucosamine 28.9% galactosamine 9.6%, and less than 0.001% labelling in the amino acids. Similarly sodium [1-14C] and [2-14C] acetate are incorporated into mucin (l2.8% of total added counts from [2-14C] acetate incorporated) into both the monosaccharide constituents and into the N-acyl substituents. [U-14C] -L-threonine (mucin 1.52% of total added counts) leads to labelling only in the peptide portion of fraction H and solely at threonine. Moreover 35SO4 2- (1.29% of total added counts incorporated) is incorporated only as ester sulphate. The incorporation of radioactivity from a variety of radioactive precursors suggests that biosynthesis of fraction H Is occurring, in contrast to the mere exchange of residues in the preformed mucoprotein.
A main aspect of the investigation has been concerned with the biochemical origin of the glycollyl residues in this mammalian tissue, Radioactivity from [2-14C] -D-glucose is incorporated by the whole tissue into both the N-glycollyl substituent and the central sugar moiety of N-glycollylneuraminic acid of mucin. However despite N-glycollylneuraminic acid being quantitatively the predominant sialic acid (N-glycollyl- to N-acetylneuraminic acid 5:3 by wt.), the ratio of radioactive labelling is 10.8:1 (as specific activities in favour of N-acetylneuraminic acid). The labelling in N-acetylneuraminic acid being comparable to that in the other monosaccharide constituents. Similar low labelling of N-glycollylneuraminic acid occurred on incubation of tissue with [2-14C] acetate, ratio of labelling of N-acetyl- to N-glycollyl- 6.6:1 (as specific activities). This low level of labelling in the N-glycollylneuraminic acid is especially evident in its main monosaccharide moiety when [2-14C]-D-glucose was used. This may be a genuine reflection of the differences in the metabolic pathways leading to these sialic acids. Alternatively it may reflect differences in pool sizes or a lower rate of biosynthesis of N-glycollylneuraminic acid.
Investigations of other possible biochemical sources of the N-glycollyl- substituent, suggest that the commonly accepted pathways of glycollic acid biosynthesis postulated for mammals, are insufficient to account for the present results. Cell-free preparations quantitatively accomplished enzyme biosynthesis of N~glycollyl- and N-acetylneuraminic acids from added N-glycollyl- and N-acetylglucosamine respectively. However, while such enzyme preparations also catalysed the formation of N-acetylglucosamine from glucosamine and acetate (or acetyl-CoA) N-glycollylation of glucosamine with a wide a variety of N-glycollyl precursors was not brought about. Fructose-6-phosphate, hydroxy- pyruvate, glycollate and chemically synthesized glycollyl-CoA all were inactive in cell-free preparations as N-glycollyl donors. In intact cells, evidence does not support the operation of these substrates as N-glycollyl- precursors, The labelling pattern of N~glycollylneuraminyl residues from [2-14C]-D-glucose and [2-14C]-acetate experiments is not compatible with fructose-6-phosphate or any other transketolase substrate being an N-glycollyl precursor. While [3-14C]-hydroxypyruvate lead to radioactivity being incorporated into galactose, fucose and to a lesser extent into hexosamines and N-acetylneuraminic acid (mucin 3.5 and 0.76% incorporation of total added counts for two experiments respectively), no incorporation into the N-glycollylneuraminic acid occurred. [U-14C] Glycine is incorporated (mucin 0.14 and 0.13% of total added counts) as glycine (or glutamic acid) and serine residues but not into the N-glycollyl residue. It is suggested that these results do not role out other possibilities such as hydroxylation of an acetyl moiety being the N-glycollyl source.
A further aspect of the work has been concerned with the inhibition of mucoprotein biosynthesis by salicylate. Salicylate (15 mM) inhibited the incorporation of [2-14C]-D-glucose (83.9%), [U-14C] -L- threonine (82%) and 35SO42- (79%) into mucin and simultaneously reduced the oxygen uptake by the tissue (595). Lower inhibitory concentrations of salicylate (3.75mM) while little effecting [U-14C] -L-threonine incorporation into mucin (3 and 6% inhibition) substantially decreased the incorporation of [2-14C]-D-glucose (48 and 41% inhibition) and 35SO42- (59 and 4O% inhibition). Analysis of mucin from inhibitions with [2-14C]-D-glucose in the presence of 3.75 mM salicylate showed that there was a large reduction in the labelling of the sialic acids (of N-acetylneuraminic acid and N-glycollylneuraminic acid 57 and 34% inhibition respectively) and hexosamines (e.g. glucosmine and galactosamine 55 and 33% inhibition respectively) while the neutral sugars were relatively unaffected (fucose and galactose 9 and 11% inhibition respectively). While fructose-6-phosphate transaminase and glucosamine-6-phosphate acetylase activities In the particle-free supernatant are relatively unaffected by salicylate, acetyl-CoA synthetase activity is markedly inhibited at salicylate levels as low as 0.235 mM (49% inhibition with 0.05 mM CoA).
In light of the above effects of salicylate on mucin biosynthesis it is suggested that the known disruptive effect of aspirin on the gastric mucosa of humans and experimental animals maybe in part due to inhibition by the drug (or its active principal salicylate) of biosynthesis of the protective mucin lining of the gut. This concept is further supported by experiments showing inhibition by 15 mM aspirin of the incorporation of [U-14C]-D-glucose into mucin (73% inhibition). Furthermore human gastric mucosa inoubated under identical conditions to the sheep mucosal tissue above, has a Qo2 of -9.9 and incorporates [U-14C]-D-glucose (15.8% of total added counts) into bound:hexosamines (20.6%), hexoses (glucose and galactose 5.7%) and facose (l4.2%). 15 mM salicylate in the incubation medium produced little observable change on the oxygen uptake by the human gastric mucosal tissue, but reduced the incorporation of [U-14C]-D-glucose into the bound sugars by 74% and this decrease was also reflected in the labelling of all the radioactive sugar constituents.
Puromyoin (0.01%) a specific inhibitor of protein biosynthesis at the ribosomal level, was found to inhibit the incorporation of L-threonine into fraction H (93.3 and 81.5% inhibition). [U-14C]-D-glucose (52.0 and 68.4% inhibition) and 35SO42- (78.9 and 76.2% inhibition) incorporation was also inhibited by puromycin but to a lesser extent than with L-threonine. The oxygen uptake of the tissue over the first 3 hours of incubation was unaffected by puromycin. Analysis of the distribution of labelling of [U-14C]-D-glucose in the puromycin samples showed that in contrast to salicylate the reduction in [U-14C]-D-glucose was reflected non-specifically in all the monosaccharide constituents e.g. for a mucoprotein sample where the decrease was 52% of total incorporation of [U-14C]-D-glucose relative to the control these were:- 56% N-acetylneuraminic acid, N-glycollylneuraminic acid 39% hexosamines 54%, fucose 42% and galactose 66%. Assuming the only effect of puromycin is on inhibition of protein synthesis in this system then these results suggest that the biosynthesis of the carbohydrate and ester sulphate moieties are closely dependant on the prior formation of a peptide precursor, and in particular sulphate incorporation is closely connected with the biosynthesis of the rest of the molecule.
Preliminary reports of this work have been published (Allen and Kent, 1966 a and b).