Glycosylation analysis and protein structure determination of murine fetal antigen 1 (mFA1)--the circulating gene product of the delta-like protein (dlk), preadipocyte factor 1 (Pref-1) and stromal-cell-derived protein 1 (SCP-1) cDNAs

By means of sequence analysis, murine fetal antigen 1 (mFA1) isolated from Mus musculus amniotic fluid was shown to be the circulating protein of the delta-like protein, stromal-cell-derived protein 1 (SCP-1) and preadipocyte factor 1 (Pref-1) gene products. The protein contains 36 cysteine residues arranged in six epidermal-growth-factor-like domains. The purification of several C-terminal peptides of varying lengths showed mFA1 to be C-terminal heterogeneous. O-linked glycosylations of the NeuNAc alpha2-3Gal beta1-3(NeuNAc alpha2-6)GalNAc type were present on all C-terminal peptides at residues Thr235, Thr244 and Thr248, although glycosylation on Thr244 was only partial. Three N-linked glycosylations were localized in mFA1 (Asn77, Asn142 and Asn151), two of which (Asn142 and Asn151) were in the unusual Asn-Xaa-Cys motif. Fucosylated biantennary complex-type and small amounts (less than 5%) of triantennary complex-type structures were identified on the glycosylated asparagine residues using sequential exoglycosidase and endoglycosidase digestions combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The presence of O-linked monosaccharides (glucose attached to Ser71, Ser193 and fucose at Thr201) was tentatively ascertained by combining Edman degradation and MALDI-MS. The results presented shows mFA1 to be the circulating heterogeneous cleavage products of the membrane-bound protein encoded by the murine cDNAs dlk, pref-1 and SCP-1.