S-layer, surface-accessible, and concanavalin A binding proteins of Methanosarcina acetivorans and Methanosarcina mazei.
The outermost cell envelope structure of many archaea and bacteria contains a proteinaceous lattice termed the surface layer or S-layer. It is typically composed of only one or two abundant, often posttranslationally modified proteins that self-assemble to form the highly organized arrays. Surprisingly, over 100 proteins were annotated to be S-layer components in the archaeal species Methanosarcina acetivorans C2A and Methanosarcina mazei Gö1, reflecting limitations of current predictions. An in vivo biotinylation methodology was devised to affinity tag surface-exposed proteins while overcoming unique challenges in working with these fragile organisms. Cells were adapted to growth under N2 fixing conditions, thus, minimizing free amines reactive to the NHS-label, and high pH media compatible with the acylation chemistry was used. A 3-phase separation procedure was employed to isolate intact, labeled cells from lysed-cell derived proteins. Streptavidin affinity enrichment followed by stringent wash conditions removed nonspecifically bound proteins. This methodology revealed S-layer proteins in M. acetivorans C2A and M. mazei Gö1 to be MA0829 and MM1976, respectively. Each was demonstrated to exist as multiple glycosylated forms using SDS-PAGE coupled with glycoprotein-specific staining, and by interaction with the lectin, Concanavalin A. A number of additional surface-exposed proteins and glycoproteins were identified and included all three subunits of the thermosome: the latter suggests that the chaperonin complex is both surface- and cytoplasmically localized. This approach provides an alternative strategy to study surface proteins in the archaea.
Francoleon
Deborah R
DR
Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
Boontheung
Pinmanee
P
Yang
Yanan
Y
Kin
UnMi
U
Ytterberg
A Jimmy
AJ
Denny
Patricia A
PA
Denny
Paul C
PC
Loo
Joseph A
JA
Gunsalus
Robert P
RP
Loo
Rachel R Ogorzalek
RR
D019843:Archaeal Proteins / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003208:Concanavalin A / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D017020:Methanosarcina / Q000378:metabolism*; D011485:Protein Binding; D020543:Proteome; D011952:Receptors, Concanavalin A / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D019843:Archaeal Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D020543:Proteome; D011952:Receptors, Concanavalin A; C055764:S-layer proteins; D003208:Concanavalin A