Determination of carbohydrate structures N-linked to soluble CD154 and characterization of the interactions of CD40 with CD154 expressed in Pichia pastoris and Chinese hamster ovary cells.
CD40-CD154 (CD40 ligand) interactions are essential for the development of protective immunity. Previous studies have described the CD40 binding site as a shallow groove formed between two monomers of CD154. However, these studies have not examined the structure or biological function of the carbohydrate on CD154. Human CD154 contains a single N-linked glycosylation site at asparagine 240. We have characterized the interactions between CD40 and soluble (s) CD154 in which sCD154 contains different types of carbohydrates. Detailed carbohydrate analysis revealed high-mannose structures on sCD154 purified from Pichia pastoris, whereas CD154 purified from Chinese hamster ovary E1A contained heterogeneous populations of complex carbohydrates. sCD154 purified from either system was trimeric, it bound to CD40 with similar affinities of 10-30 nM, and it functionally induced CD69 and CD95 expression on primary B cells. Together, these results indicate that the presence of varied types of N-linked glycans on asparagine 240 of CD154 does not play a significant role in the CD40-CD154 interactions.
Khandekar
S S
SS
Department of Protein Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA.
Silverman
C
C
Wells-Marani
J
J
Bacon
A M
AM
Birrell
H
H
Brigham-Burke
M
M
DeMarini
D J
DJ
Jonak
Z L
ZL
Camilleri
P
P
Fishman-Lobell
J
J
D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001402:B-Lymphocytes / Q000276:immunology; D019013:CD40 Antigens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D023201:CD40 Ligand / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016466:CHO Cells; D002236:Carbohydrate Conformation; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006224:Cricetinae; D006801:Humans; D008358:Mannose / Q000737:chemistry / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D019013:CD40 Antigens; D002241:Carbohydrates; D011994:Recombinant Proteins; D023201:CD40 Ligand; D001216:Asparagine; D008358:Mannose