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A mouse myeloma tumor was used as a model system to study the biochemical steps involved in the incorporation of mannose into glycoproteins. This tumor, MOPC-46B, synthesizes a K-type immunoglobulin light chain (K-46) which is a glycoprotein with a single oligosaccharide side chain containing mannose as one of its constituent sugars. MOPC-46B microsomal preparations contain enzymes which transfer mannose from the sugar nucleotide, GDPmannose, to endogenous lipid and protein acceptors. Formation of the mannolipid proceeds by the reversible transfer of mannose from GDP-mannose to an endogenous phospholipid. The mannolipid was purified and characterized by chemical methods and mass spectrometry as a mannosyl-monophosphoryl- dihydropolyisoprenol, containing at least 18 isoprene units, one of which is saturated. The mannolipid was implicated as an intermediate in the in vitro mannosylation of endogenous protein acceptors by three kinds of experiments. (a) Incorporation of [l%]mannose into protein was observed after the initial substrate, GDP-mannose, had been destroyed by sugar nucleotide hydrolases associated with the microsomal preparations. The continued increase in radioactivity in the protein fraction occurred concomitantly with a loss of radioactivity from the mannolipid fraction. (b) I ncorporation of [14C]mannose into both lipid and protein was inhibited by EDTA added at zero time. However, addition of EDTA after mannolipid synthesis had occurred resulted in cessation of mannolipid formation but continued incorporation of mannose into protein to an extent proportional to the amount of mannolipid originally formed. The increase in radioactivity in protein was again accompanied by a loss of radioactivity from the mannolipid. (c) When microsomes were pulsed briefly with GDP-[14C]mannose, which was then chased by a large excess of unlabeled GDP-mannose, incorporation of [‘XZ]mannose into lipid ceased immediately with the chase, while incorporation into protein continued afterwards to an extent proportional to the amount of mannolipid formed prior to the chase. Evidence that the mannolipid could function as a donor of mannose residues to protein was obtained by demonstrating that microsomes catalyze the transfer of [*4C]mannose from exogenously supplied mannolipid to endogenous protein acceptors. The amount of mannose transferred to protein was proportional to both microsomal protein and lipid concentrations. In addition, the amount of mannose transferred to protein from exogenous mannolipid is comparable to that incorporated from an equivalent amount of mannolipid generated endogenously from GDP-mannose. Gel filtration profiles of the [14C]mannose-containing protein formed in this system are essentially identical regardless of whether GDP-mannose or mannolipid is used as substrate. In both cases the radioactive protein fractionates in a manner similar to authentic K-46 (mol wt 24,000). The mannose-containing protein formed from either GDPmannose or mannolipid was degraded sequentially by Pronase and subtilisin. The products formed from either substrate appeared to be identical and exhibited chromatographic and electrophoretic characteristics of glycopeptides. It was concluded that mammalian microsomal preparations contain an endogenous phospholipid, characterized as a dihydropolyisoprenol-monophosphate, which serves as an acceptor of mannose from GDP-mannose, resulting in the formation of mannosyl-monophosphoryl-dihydropolyisoprenol, and that this mannolipid serves as a glycosyl donor for transfer of mannose residues to endogenous protein acceptors. The evidence indicates that the mannolipid is an essential intermediate in the in vitro transfer of mannose from GDP-mannose to protein.

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