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 prote in of the delta-like protein, stromal-cell-derived protein 1 (SCP-1) a nd preadipocyte factor 1 (Pref-1) gene products. The protein contains 36 cysteine residues arranged in six epidermal-growth-factor-like doma ins. The purification of several C-terminal peptides of varying length s showed mFA1 to be C-terminal heterogeneous. O-linked glycosylations of the NeuNAc alpha 2-3Gal beta 1-3(NeuNAc alpha 2-6)GalNAc type were present on all C-terminal peptides at, residues Thr235, Thr244 and Thr 248, 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. F ucosylated biantennary complex-type and small amounts (less than 5%) o f triantennary complex-type structures were identified on the glycosyl ated asparagine residues using sequential exoglycosidase and endoglyco sidase digestions combined with matrix-assisted laser desorption/ioniz ation mass spectrometry (MALDI-MS). The presence of O-linked monosacch arides (glucose attached to Ser71, Ser193 and fucose at Thr201) was te ntatively ascertained by combining Edman degradation and MALDI-MS. The results presented shows mFA1 to be the circulating heterogeneous clea vage products of the membrane-bound protein encoded by the murine cDNA s dlk, pref-1 and SCP-1.