Structures of disease-specific serum alpha-fetoprotein isoforms.

Alpha-fetoprotein (AFP) is widely used as a serological marker in the diagnosis of hepatocellular carcinoma (HCC) and non-seminomatous germ cell tumours (NSGCT). By application of isoelectric focusing (IEF) disease-specific AFP isoforms can be identified. Three major bands are apparent: + 1 (associated with 'benign' liver disease), + II (associated with HCC) and +III (associated with NSGCT). Recently, we have characterized the predominant glycans of human serum AFP and now report the application of these findings and electrospray ionization-mass spectrometry (ESI-MS) to the determination of the glycan composition of the isoforms present in the sera of 12 patients with HCC and of one patient with NSGCT. ESI-MS allowed simultaneous identification of various AFP glycoforms in purified serum AFP. Seven glycoforms were identified, but with different abundance in the sera of the HCC patients, whereas six glycoforms were identified in the serum from the NSGCT patient. The glycan structures of these glycoforms were deduced from their observed masses. AFP glycoforms carrying a single biantennary complex-type N -glycan appeared as the predominant glycoforms, whereas those carrying both N -glycan and O -glycan appeared as minor glycoforms. Correlation between the abundance of the AFP glycoforms and the IEF band intensity suggested that different degrees in sialylation cause the formation of isoforms. This contention was subsequently supported by the ESI-MS and kinetic in vitro desialylation studies on purified Bands + l and + lI AFPs. Our findings indicate that HCC-associated isoforms (Band + II) represent a group of glycoproteins whose carbohydrate structures are all characterized by being mono-sialylated, whereas those associated with benign liver disease and NSGCT are di- and a-sialo species, respectively. Knowledge of the structure of the tumour-specific isoforms should form an important basis for clinically useful assays.