Bovine submandibular gland mucin

Neu5Ac or Neu5Gc with 0-acetyl groups at positions 7, 8, and 9 may be prepared from bovine submandibular gland mucin. Isolation of the crude mucous glycoprotein before hydrolysis is advisable to improve the resolution of sialic acids... 

In contrast, in bovine submandibular gland mucin, neither of the initial periods of oxidation abolished subsequent PAS reactivity although the PAS reactivity was somewhat weaker in sections exposed to an initial period of 30 minutes. We interpret these results as evidence for the presence of 9-0-acyl sialic acids although not all such acids can be demonstrated by such a method since presumably some oxidation of the 9-0-acyl sialic acids will occur within the initial period of oxidation. 

Histochemical demonstration of most of the O-acetylated sialic acids is possible because substituents on the side chain of Neu hinder periodate oxidation of this part of the molecule to an extent dependent on the number and position of the O-acetyl groups already mentioned. Correspondingly, removal of these ester groups by alkaline treatment (0.5% KOH in 70% ethanol91) may increase the staining reaction of a sialic acid. For example, the presence of O-acylated sialic acids has been demonstrated in colonic, epithelial mucin of man and various mammals (summarized in Ref. 91), in healthy and diseased, human small-intestine,188-190 in bovine submandibular gland,182 in mouse and rat erythrocyte-membranes,191 and in human lymphocytes.192... 

To investigate the latter possibility, sections of bovine submandibular gland and human colon were oxidized for periods of either 10 or 30 minutes and were then reduced with sodium borohydride. The sections were then reoxidized with periodate for various periods and then stained with Schiff reagent. The results obtained showed that when human colonic mucins were oxidized for an initial period of 10 minutes, some PAS reactivity could be regenerated by a second oxidation period of 60 minutes. When however, the initial oxidation period was extended to 30 minutes, no further PAS reactivity could be generated by a second period of oxidation. These results could be interpreted in two ways either they were the result of the incomplete oxidation of a relatively small number of 9-0-acyl sialic acids or the failure to oxidize other tissue diols. The probability of the latter was lessened by the observation that the oxidation of the crypt mucins of rabbit small intestine which do not contain 0-acylated sialic acids was apparently complete in 10 minutes. 

Figure 3. Two models for the O-acetylation of Sia in Golgi-membranes of bovine submandibular glands. A, as CMP-glycoside, before the transfer onto nascent glycoconjugates (mucin) and B, after this transfer. OAT, sialate-7(9)-0-acetyltransferase ST, sialyltransferase M, O-acetylisomerase. For further details see the text.

The sialic acids whose structures were recently questioned by the above mass spectral data are indicated in Table I according to both the original and new assignments. In support of the revised structures, synthetic 9-0-acetyl derivatives were shown to react slowly with periodate comparable to the naturally occurring sialic acids (Haverkamp et al., 1975). Thus far no reports have appeared concerning the location of acetyl groups on the )V-acetyl-tri-0-acetylneuraminic acid (7) obtaind from bovine submaxillary mucin. Very recently a new sialic acid was discovered (Schauer et al., 1976) in bovine submandibular gland N-aceytl-9-O-L-lactylneuraminic acid (II). 

---