The Heterogeneity of Human Gc-globulin*

SUMMARY Gc-globulin or group-specific component, also known as the vitamin D-binding protein, was investigated by the combined use of electrofocusing and immunofixa-Con. Serum of the Gc 2-2 type was found to contain a single protein band whereas serum of the Gc l-l type shows two bands with a lower isoelectric point. The Gc l-2 type contains all three bands known as Gc-2 (p1 &lo), Gc-1Slow (p1 5.03), and Gc-1Fast (p1 4.95). Each apoprotein shows an anodal shift of about 0.07 pH unit after incubation with an excess of 25-hydroxycholecal- ciferol. After treatment with sialidase Gc-1Fast focuses in the position of Gc-lSlow, whereas the position of Gc- 2 remains unchanged.


SUMMARY
Gc-globulin or group-specific component, also known as the vitamin D-binding protein, was investigated by the combined use of electrofocusing and immunofixa-Con. Serum of the Gc 2-2 type was found to contain a single protein band whereas serum of the Gc l-l type shows two bands with a lower isoelectric point. The Gc l-2 type contains all three bands known as Gc-2 (p1 &lo), Gc-1Slow (p1 5.03), and Gc-1Fast (p1 4.95). Each apoprotein shows an anodal shift of about 0.07 pH unit after incubation with an excess of 25-hydroxycholecalciferol. After treatment with sialidase Gc-1Fast focuses in the position of Gc-lSlow, whereas the position of Gc-2 remains unchanged.
Human Gc-globulin,' discovered in 1959 by Hirschfeld (l), shows three common phenotypes which are determined by a pair of co-dominant autosomal alleles (Gc' and Gc'). Serum of people homozygous for Cc' (Gc l-1) contains Gc migrating electrophoretically as a doublet known as Gc-1Fast and Gc-lSlow, whereas serum from people homozygous for Gc2 (Gc 2-2) shows a single Gc-2 band. Serum of heterozygotes (Gc l-2) contains all three bands. The finding that this plasma protein has binding properties for 25-OH-D3 (2)(3)(4)(5) (6), it was found that more than 90% of the sialic acid was released.
Measurement of Gcglobulin by single radial immunodiffusion (7) indicated that sialidase treatment did not alter the Gc-globulin concentration.

AND DISCUSSION
Phenotyping of Human Gc-globulin-Different serum samples of known Gc-globulin composition were focused on polyacrylamide gels, followed by immunofixation.
Three different patterns were obtained, having one, two, or three protein bands, corresponding to Gc 2-2, Cc l-l, and Gc l-2, respec- as present in serum, and the corresponding holoprotein, obtained after incubation with a 25-fold molar excess (10 PM) of 25-OH-Da, differ by 0.07 pH unit. Nonsaturating amounts of 25-OH-D3 produced two Gc bands, corresponding to the isoelectric position of the apo-and holoprotein. This is clearly demonstrated when a serum sample of the Gc 2-2 type is focused after addition of increasing amounts of 25-OH-D3 (Fig. lc). Equimolar concentrations of Gc-globulin and 25-OH-D3 result only in about 50% conversion, probably due to lower affinity at this pH.
In another experiment, the influence of a 25-fold molar excess (10 pM) of 24,25-dihydroxycholecalciferol, la,25-dihydroxycholecalciferol, and vitamin DB was investigated. The anodic shift was observed with 24,2&dihydroxycholecalciferol, partially with la,25-dihydroxycholecalciferol but not with vitamin DS. This probably reflects differences in affinity of these vitamin D metabolites for Gc-globulin.
A similar anodic shift of the vitamin D-binding protein of rat serum was observed by the addition of excess 25-OH-Dz.
The molecular basis of the vitamin D-induced charge difference remains speculative and could be explained either by a conformational change or by the burying of (an) ionizable group(s) of the binding protein. A similar phenomenon has been described for the desialylated form of human transcortin which shows an anodic shift of 0.2 pH unit as the result of cortisol binding (10). The heterogeneity induced by 25-OH-D3 binding is unlikely to be detected in normal human serum since Cc-globulin circulates mainly as an apoprotein (7). However, in vitamin D-treated patients, a supplementary Gc band will be detected, as shown in Fig. 1C and recently described by Brissenden and Cox (11). This vitamin D-induced charge difference also implies that autoradiography of 25-OH-[14C]D3-labeled serum will detect the position of the holoprotein which differs 0.07 pH unit with the pattern detected by immunofixation.
The present results also indicate that, as previously stated (lo), trace amounts of radioactive ligands are not always good markers for the isolation of a binding Gc l-l 2-2 l-2 l-l 2-2 1-2 globulin, as measured immunochemically, it is inferred that Gc-1Fast focuses in the position of Gc-1Slow. Addition of 25-OH-D3 in 25-fold molar excess (10 pM) to sialidase-treated samples results in an anodic shift of about 0.07 pH unit of all components, showing that this treatment does not alter the vitamin D-binding properties of Gc-globulin. Since Gc-1Fast and Gc-1Slow co-focus after sialidase treatment, they differ certainly in their carbohydrate composition. Whether they differ in amino acid composition remains to be determined. Acknowledgments-The technical assistance of Mrs. K. Vandecauter-Deruddere is gratefully appreciated. We thank Roussel-Uclaf (Paris, France) and Hoffmann-La Roche (Basle, Switzerland) for their generous gift of 25-hydroxycholecalciferol, la, 25-dihydroxycholecalciferol, and 24,2&dihydroxycholecalciferol.