Phosphorous carbohydrates on chimeric CD22, CD44 and CD62L.
There are few examples of phosphorous carbohydrates described in higher animals. Here we have used recombinant molecules where the extracellular part of membrane receptors have been fused with the Fc part of human IgG (Rg-chimeras) to look at the prevalence of phosphorous carbohydrates. Also chimeras of a few hormones were used in this study. Thirteen Rg constructs were transfected into Cos cells and labelled with [32P]orthophosphate in phosphor deficient media. These Rg molecules were subsequently purified on protein A-Sepharose and submitted to SDS-PAGE and radiography. CD22Rg, CD62L-Rg and CD44Rg were all labelled very strongly with 32P. From CD22Rg and CD62L-Rg the label could be easily removed by N-glycosidase F, but the 32P label on CD44Rg was resistant to N-glycosidase treatment. However, after treatment with O-glycosidase combined with sialidase, CD44Rg retained only a fraction of the 32P. Weakly phosphorylated Rg molecules were CD62E-Rg and CD7Rg. However, CD7Rg did not seem to loose the label, only shift position after N-glycosidase treatment and CD62E-Rg did neither shift position nor loose any 32P label after the N-glycosidase treatment. Negative chimeras were CD40Rg, CD33Rg, Lamp-1Rg, CD34Rg, ICAM-1Rg, TNF alpha Rg, CD19Rg and CD5Rg. The phosphate label of CD22Rg was completely removed after ALP treatment and in CD44Rg most of the label was removed. ALP is not supposed to cleave phosphodiesterbonds leading to the conclusion that the phosphor is likely present in the form of phosphomonoesters. This result is interesting as alkaline phosphatase (ALP) is common on the outer cellsurface of many cell types and might interact with phosphorous carbohydrates on membrane proteins. The membrane from of CD22 was also transfected into Cos cells and labelled with 32P in phosphate free media. Subsequently the cells were lysed and CD22 affinity purified and analysed by SDS-PAGE and radiography. Under these conditions CD22 was shown to be 32P labelled, but only 20% of the label was removed by N-glycosidase F treatment. These results do not show that CD22, CD44 and CD62L are phosphorylated on carbohydrates under more physiological circumstances, but they do show that phosphorous carbohydrates might be more common in higher animals than what was been reported.
Jönsson
G
G
Immunology Department, University of Stockholm, Sweden.
Gelius
E
E
Braesch-Andersen
S
S
D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics; D000944:Antigens, Differentiation, B-Lymphocyte / Q000737:chemistry / Q000235:genetics; D002241:Carbohydrates / Q000737:chemistry*; D015815:Cell Adhesion Molecules; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D018960:Hyaluronan Receptors / Q000737:chemistry / Q000235:genetics; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics; D019041:L-Selectin / Q000737:chemistry / Q000235:genetics; D037102:Lectins; D010758:Phosphorus / Q000032:analysis*; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D051918:Sialic Acid Binding Ig-like Lectin 2
D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; C497867:CD22 protein, human; D002241:Carbohydrates; D015815:Cell Adhesion Molecules; D006023:Glycoproteins; D018960:Hyaluronan Receptors; D007141:Immunoglobulin Fc Fragments; D037102:Lectins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D051918:Sialic Acid Binding Ig-like Lectin 2; D019041:L-Selectin; D010758:Phosphorus