Resolution and in vitro glycosylation of membrane glycoproteins in Micrococcus luteus (lysodeikticus)

In contrast to the situation in eukaryotic cells, the membrane-associated carbohydrate of bacteria occurs largely in molecular forms (e.g. lipopolysaccharides and lipoteichoic acids) other than glycoproteins [1]. Notable exceptions are found in the Archaebacteria where at least two species, viz. Halobacterium salinarium [2] and Thermoplasma acidophilum [3] possess glycoproteins as major constituents of their cell envelope. A glycoprotein has also been isolated from the membranes of Mycoplasma pneumoniae [4]. However, the evidence for membrane glycoproteins in other bacteria is more tenuous and controversial [1,2,5-11]. Our current interest in bacterial glycoproteins stems from a number of observations made on the model Gram-posit ive organism Micrococcus luteus (lysodeikticus). Membranes from this bacterium are unusually rich in mannose, much of which is in the form of a well characterized succinylated lipomannan [ 12-15]. However, there is some evidence from crossed immunoaffinoelectrophoresis experiments conducted with the lectin concanavalin A [16] and from periodic acid-Schiff (PAS) stains performed on sodium dodecyl sulphate (SDS)polyacrylamide gels [12] for the presence of additional glycosylated species. Furthermore, Mufioz and his coworkers [5,8,9] have presented evidence, albeit of a controversial nature [10,11], which suggests that the F~. ATPase of this organism is a glycoprotein. Finally, we had certain reservations about the precise chemical nature of the mannosyl acceptors in the M. luteus "mannan synthetase" assay [13]. High resolution analysis of this material has not been presented and it is less than clear that it corresponds to the succinylated l ipomannan as proposed [ 13]. In the present communication we establish that, in the presence of GDP[14C]mannose, membranes of M. luteus are capable of catalyzing the glycosylation of a number of endogenous proteins. Furthermore, we establish that the "mannan synthetase" assay of Scher and Lennarz [13] monitors this process in large part.