Biochemical characterization and crystalization of human Zn-alpha2-glycoprotein, a soluble class I major histocompatibility complex homolog

Abstract

Zn-alpha2-glycoprotein (ZAG) is a 41-kDa soluble protein that is present in most bodily fluids. In addition, ZAG accumulates in fluids from breast cysts and in 40% of breast carcinomas, which suggests that ZAG plays a role in the development of breast diseases. However, the function of ZAG under physiological and cancerous conditions remains unknown. Because ZAG shares 30-40% sequence identity with the heavy chains of class I major histocompatibility complex (MHC) proteins, we compared the biochemical properties of ZAG with those of classical class I MHC molecules. We purified human ZAG from breast cyst fluid and serum and produced a panel of anti-ZAG monoclonal antibodies. Binding assays and acid elution experiments revealed that, in contrast to class I MHC proteins, ZAG does not bind peptides or the class I light chain, beta2-microglobulin (beta2m). Nevertheless, CD studies indicated that ZAG is thermally stable in the absence of bound peptide or associated beta2m, as opposed to class I MHC molecules, which require the presence of both beta2m and peptides for stability. These data indicate that the function of ZAG has diverged from the peptide presentation and T-cell interaction functions of class I molecules. To gain insight into the function of ZAG and to compare the three-dimensional structures of ZAG and class I MHC molecules, we produced ZAG crystals that diffract beyond 2.7 A and have initiated an x-ray structure determination.

Figure 1

PAGE analysis of ZAG. Aliquots of ZAG purified from breast cyst fluid or serum were analyzed by SDS/10–15% PAGE and native/8–20% PAGE.

Figure 2

Chromatographic and immunoprecipitation analyses confirm that ZAG does not bind β₂m. (A) β₂m (Top), ZAG (Middle), and a mixture of ZAG incubated with β₂m (Bottom) were analyzed by size exclusion chromatography. (Inset) SDS/13% PAGE analysis of the peaks in the bottom panel. (B) Aliquots of ZAG, serum, and β₂m were immunoprecipitated with either anti-ZAG or anti-β₂m antibodies. Precipitated proteins were analyzed by Western blot using antisera against either ZAG or β₂m.

Figure 3

HPLC analysis of acid eluates from K^d, ZAG, and a control with no protein. All peaks in the ZAG eluate were subjected to N-terminal sequencing and mass spectrometry analysis. No peptides were detected.

Figure 4

Thermal denaturation profile of serum ZAG. The CD signal at 223 nm is plotted as molar ellipticity per mean residue after smoothing. Similar profiles were obtained using ZAG isolated from cyst fluid. Thermal denaturation studies were repeated after denaturation and dialysis to remove any potential ZAG ligands without any effect on the denaturation profile.