Mannoprotein biosynthesis

In this paper I intend to summarize what we know about yeast mannoprotein structure, what features of the biosynthetic pathway have been defined, how genetic techniques have been useful in gaining this information, and what is known about the genetic control of mannoprotein biosynthesis. 

Figure 5. Postulated glycosylation pathway in mannoprotein biosynthesis. The core unit attached to asparagine is derived from Dolichol-Pz-GNAc—Many, where y is about 15, whereas the first mannose attached to serine is derived from Doli-chol-P—Man. In the above figure, x is about 3 to 5.

Several levels of control must be involved in mannoprotein biosynthesis (Table l). Because these are glycoproteins, the... 

The second level of control of mannoprotein biosynthesis involves the glycosyltransferases that construct the precursors and add them to the protein. We have already seen that the distribution of such enzyme activities in the wild population of Sacdha-myces species varies in a fashion reminiscent of the enzymes... 

The study of the biosynthesis of D-mannoproteins in other fungi is less developed. It has already been mentioned that the participation of Man-P-Dol as the glycosyl donor in O-glycosylation was demonstrated in Neuro-spora crassa97 and in Fusarium solani f. pisi.56 In the latter, the lipid acceptor was identified as C90- to C110-dolichols. In Hansenula species, O-glycosyla-tion follows the same pathway that is D-mannose linked to L-serine or L-threonine is incorporated by way of Man-P-Dol, and further elongation... 

The biosynthesis in yeast of two enzymes that are D-mannoproteins has been studied. A membrane-associated isozyme of invertase (EC 3.2.1.26) has been shown to be a precursor of the external invertase.190 Its molecular weight, as determined by SDS-poly(acrylamide) gel electrophoresis, is 50,000, that is, smaller than that of the external invertase, and it correlates well with the presence of only the inner-core sugars of the final form. It is strictly bound to membranes, possibly those of the endoplasmic reticulum, and it can be completely split191 by endo-/3-N-acetylglucosaminidase H (EC 3.2.1.30). The addition of tunicamycin, which specifically inhibits formation of d-GIcNAc-PP-DoI, inhibits synthesis of external invertase, as well as further formation of the membrane-associated form, which completely disappears after addition of the antibiotic.190 In these aspects, the synthesis of this extracellular enzyme follows the pathway for secreted glycoproteins in animal systems. 

Figure 8. Chromosome locations of genetic loci concerned with biosynthesis of the carbohydrate part of S. cerevisiae mannoprotein. The circles indicate the...

The mannan is in the form of a mannoprotein, with large blocks of branching mannan attached by V-glycosidic links to asparagine and smaller chains linked by the mannosyl-serine bond, which seems peculiar to fungi (see Chapters 1 and 4 for details of the structure and its biosynthesis). ITie polypeptide part of the mannoprotein is cross-linked in the cell wall by way of disulphide bridges, while the mannan blocks interlink by way of phosphate bridges. In the model of Kidby and Davies (1970) these cross-links lead to the... 

Carboxypeptidases.—A study of the biosynthesis of yeast carboxypeptidase Y (a mannoprotein) has revealed that a membrane fraction contains a precursor of the enzyme, which, although it has a higher molecular weight than the enzyme, is not a complex of the enzyme and its specific inhibitor. ... 

Once the structure of mannan was established to be manno-protein(s) we studied its biosynthesis by following the formation of both the peptide and carbohydrate moieties. In these studies one of the most powerful techniques is the dissection of a metabolic route by inhibiting specific reactions with antimetabolites. In the case of mannoproteins the two moieties are quite different and are polymerized by different biosynthetic routes. We inhibited protein formation with the antibiotic cycloheximide which freezes translation of the genetic message at the level of polysomes. 

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