Glycosylation of homologous immunodominant proteins of Ehrlichia chaffeensis and Ehrlichia canis

The glycoprotein genes of Ehrlichia chaffeensis (1,644 bp) and Ehrlichia ca nis (2,064 bp) encode proteins of 548 to 688 amino acids with predicted mol ecular masses of only 61 and 73 kDa but with electrophoretic mobilities of 120 kDa (P120) and 140 kDa (P140), respectively. The 120-kDa protein gene o f E. chaffeensis contains four identical 240-bp tandem repeat units, and th e 140-kDa protein gene of E. canis has 14 nearly identical, tandemly arrang ed 108-bp repeat units. Conserved serine-rich motifs identified in the repe at units of P120 and P140 were also found in the repeat units of the human granulocytotropic ehrlichiosis agent 130-kDa protein and of the fimbria-ass ociated adhesin protein Fap1 of Streptococcus parasanguis. Nearly the entir e (99%) E. chaffeensis P120 gene (1,616 bp), the 14-repeat region (78%) of the E. canis P140 gene (1,620 bp), and a 2-repeat region from the E. chaffe ensis P120 gene (520 bp) were expressed in Escherichia coli. The recombinan t proteins exhibited molecular masses ranging from 1.6 to 2 times larger th an those predicted by the amino acid sequences. Antibodies against the reco mbinant proteins reacted with E. chaffeensis P120 and E. canis P140, respec tively. Carbohydrate was detected on the E. chaffeensis and E. canis recomb inant proteins, including the two-repeat polypeptide region of E. chaffeens is P120. A carbohydrate compositional analysis identified glucose, galactos e, and xylose on the recombinant proteins. The presence of only one site fo r N-linked (Asn-Xaa-Ser/Thr) glycosylation, a lack of effect of N-glycosida se F, the presence of 70 and 126 Ser/Thr glycosylation sites in the repeat regions of P120 and P140, respectively, and a high molar ratio of carbohydr ate to protein suggest that the glycans may be O linked.