Binding properties of the galactose-detecting lectin Pseudomonas aeruginosa agglutinin (PA-IL) to skeletal muscle fibres. Quantitative precipitation and precipitation inhibition assays.
Pseudomonas aeruginosa agglutinin (PA-IL) staining and the influence of various carbohydrates on lectin binding to muscle sections were investigated quantitatively using a scanning and integrating microspectrophotometre. A strong dose-dependent inhibition of PA-IL staining in the sections was recorded with galabiose (Galalpha1-4Gal) while lactose (Galbeta1-4Glc) had no inhibitory effect. The affinity of PA-IL to Galalpha1 carbohydrates was studied by ELISA using immobilized glycoconjugates in which Galalpha1 glycans were attached to bovine serum albumin or ceramide. PA-IL exhibited strong binding to both simple glycoconjugates having a single Galalpha moiety and to di- and trisaccharides with terminal Galalpha1 at the non-reducing end. In all cross-sectioned muscle fibres incubated with PA-IL, the staining was present as a honeycomb-shaped network through the entire cytoplasm. Further, a dense punctuate staining could be shown in most fibres. A similar staining pattern was noticed after incubation with a monoclonal antibody against ryanodine receptors and with biotinylated ryanodine suggesting that the network could represent the sarcoplasmic reticulum. Further, Western blots of a sarcoplasmic reticulum preparation showed multiple bands after incubation with PA-IL. It may therefore be proposed that glycoconjugates carrying terminal Galalpha1 show affinity for PA-IL and are located to the sarcoplasmic reticulum.
Kirkeby
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S
Department of Oral Function and Physiology, Faculty of Health Sciences, University of Copenhagen, Denmark.
Høyer
P E
PE
D018829:Adhesins, Bacterial / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002415:Cats; D011232:Chemical Precipitation; D004797:Enzyme-Linked Immunosorbent Assay; D005690:Galactose / Q000378:metabolism; D007150:Immunohistochemistry; D037102:Lectins / Q000378:metabolism; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000302:isolation & purification
D018829:Adhesins, Bacterial; D037102:Lectins; D019837:Ryanodine Receptor Calcium Release Channel; C038692:adhesin, Pseudomonas; D005690:Galactose