David L. Emerson

Gc (vitamin D binding protein) binds to cytoplasm of all human lymphocytes and is expressed on B-cell membranes☆

Peripheral blood lymphocytes were examined immunohistologically for evidence of interactions with Gc protein, a major vitamin D binding protein in serum. In the cytoplasm, binding sites for purified Gc were readily detectable in all cells, and these sites were only partially occupied by Gc. In contrast, on the membrane of viable cells, there was negligible evidence of binding of either the apo- or holoform of Gc protein, but substantial quantities of firmly bound immunoreactive endogenous Gc were detected. Separation experiments and double-label fluorescence with antisera recognizing defined phenotypic markers showed immunoreactive membrane Gc on 30-40% of unfractionated mononuclear cells and greater than 95% of monocytes or B cells. Only 5-8% of T cells were similarly reactive; these were not apparently confined to any given subset. Extraction of unfractionated cells with 6 M urea or solubilization in Nonidet P-40 released immunoreactive Gc protein, with physicochemical properties indistinguishable from those of Gc purified from serum (apparent MW 56K; pI 4.8-5.1). These findings indicate that membrane Gc may represent another surface immunofluorescence marker for B cells, and may play a role in immunocyte function.

Evidence of Increased Gc: Actin Complexes in Pregnant Serum: A Possible Result of Trophoblast Embolism

ABSTRACT: The molecular configuration of group‐specific component Gc protein in sera from pregnant and nonpregnant individuals was compared by analytical isoelectric focusing and by print immunofixation in conjunction with known standards of Gc:actin and Gc:vitamin D3 complexes. These studies revealed that while complexes of Gc with actin and with vitamin D were detectable in small amounts in nonpregnant sera, much larger quantities of both types of complexes were consistently visualized in pregnancy. In addition, when actin was added to pregnant sera containing Gc:vitamin D3 complexes, a third anodal complex was revealed which presented the molecular configuration of actin: Gc: vitamin D3. These results demonstrate that Gc:actin complexes may be present under physiological circumstances in the circulation. Since large amounts of trophoblast enter the maternal circulation during both normal and abnormal human pregnancy, experiments were undertaken that showed that actin was released from isolated trophoblast membranes and also upon lysis of other viable cells under physiological conditions similar to those obtained in serum, and such actin complexed rapidly with Gc. Although the effects of this phenomenon upon the immunobiology of pregnancy are unknown, these findings are consistent with the concept that Gc protein may exert a “scavenger” function in mopping up actin released from damaged cells.

Accurate quantitation of native Gc in serum and estimation of endogenous Gc : G-actin complexes by rocket immunoelectrophoresis

Complex formation between purified Gc and G-actin caused increased rocket height on immunoelectrophoresis with monospecific Gc antiserum, and artifactually high calculated Gc levels. The increase in rocket height varied in log: linear fashion with the amount of G-actin present, up to a plateau attained at equimolarity. The raw Gc values could therefore be corrected to within +/- 10% of known levels by addition of excess G-actin and use of standard plots obtained with Gc after saturation with G-actin. This also allowed quantitation of the percentage of Gc complexed with G-actin. In subsequent studies of whole human sera, comparison of normal controls with pregnant subjects and patients with liver disease showed evidence of differences both in absolute quantities of Gc and the relative proportion circulating as complex with G-actin. This appeared to be due to increased release of cellular actin into the extracellular space. These results show that rocket immunoelectrophoresis can be modified to provide accurate Gc levels, and also information concerning different molecular forms of this protein.

Western blot analysis of serum antibody reactivity with human melanoma cell antigens in alopecia areata and vitiligo

Antibody reactivity to melanocyte-derived cells was investigated in patients with alopecia areata or totalis by use of Western blot analysis of detergent-solubilized membrane antigens of a human melanoma cell line, M14. Reactivity was detected in the sera of 9 of 27 alopecia areata or totalis patients, 8 of 13 vitiligo patients, and 6 of 24 normal control subjects. Significant differences between patient and control sera were found in the number and distribution of antibody specificities detected. In vitiligo sera, there was an increased prevalence of reactivity to a melanoma antigen of 52,000 mol wt. In contrast, the predominant specificities in alopecia areata sera were for antigens of 74,500 and 70,800 mol wt, and the majority of positive sera were from patients with total hair loss. These findings suggest that autoreactivity to pigmented cells occurs in certain patients with alopecia areata or totalis.

Placental Alkaline Phosphatase Is a Major Specificity in Antisera Raised to Human Trophoblast Membranes

ABSTRACT: The antigens recognized by heteroantisera raised to human trophoblast membrane were studied on a variety of normal tissues and certain neoplastic cell lines, both by immunohistological and immunoprecipitation techniques. Native antisera reacted with all tissues examined, but after absorption with normal human serum and lyophilized normal liver, reactivity with normal tissues was restricted to the trophoblast membrane, endocervix, and mitogen‐ and antigen‐stimulated lymphocytes. Several membrane components were precipitated from trophoblast and activated lymphocytes by native antisera, whereas after absorption a single radioactive peak of MW 62,000 was obtained. HeLa, Chang, AV3, and HEp‐2 cell lines were also positive by immunofluorescence with the absorbed antisera, and a single molecular species was again precipitated. Additional enzymatic studies both of immunoprecipitated material and of the tissue of origin provided evidence that this species was the placental isoenzyme of alkaline phosphatase. These results indicate that the major specificity in absorbed trophoblast antisera is the heat‐stable placental isoenzyme of alkaline phosphatase. Furthermore, since similar material appears to be present on other normal and transformed tissues, this isoenzyme may not therefore, despite previous claims, be truly trophoblast‐specific.

Studies of the Normal Human Placental Syncytiotrophoblast Membrane: A Combined Immunological and Physicochemical Approach*

ABSTRACT: In view of the importance of the syncytiotrophoblast in the immunobiology of human pregnancy, the composition of this interface between fetus and mother was further studied by a combination of physicochemical and immunological approaches. Trophoblast membranes were solubilized using three classes of detergents: zwitterionic (sulfobetaine14), non‐ionic (Triton X‐100), and anionic (deoxycholate). Quantitative studies of protein released demonstrated the importance of dispersion of membranes into detergent, and optimum solubilization was then obtained at detergent:protein ratios of 1.3:1 for sulfobetaine, 2.5:1 for Triton X‐100, and 4.4:1 for deoxycholate. Analysis of chaotrope‐treated detergent‐solubilized membrane components was performed by preparative isoelectric focusing followed by combined physicochemical and immunological methods. The results revealed three major proteins which were identified as placental alkaline phosphatase, albumin, and transferrin. Certain additional low molecular weight proteins were also evident; one of these components displayed physicochemical properties similar to those of actin, but none appeared to be recognized by conventional heteroantisera raised to trophoblast membrane. These findings and the results of further immunological analysis by affinity chromatography are consistent with the concept that human trophoblast may express limited immunogenicity due in part to the presence of large amounts of absorbed maternal serum components and limited expression of fetal proteins.

Gc (vitamin D-binding protein) binds the 33.5 K tryptic fragment of actin

Limited proteolysis of G-actin was performed with trypsin and chymotrypsin to compare the binding sites for Gc and DNase. DNase I bound to the N-terminal area corresponding to the major cleavage site on G-actin (residues 62-68) and inhibited proteolysis, but did not bind the 33.5K C-terminal fragment (G-actin33.5) generated. In contrast, Gc did not exert any inhibitory effect upon proteolysis of the intact native G-actin42.0 molecule, although its presence protected G-actin33.5 from further proteolysis. This was shown by gel filtration to be due to the formation of complexes between Gc and G-actin33.5.

Interaction of α2-hs-glycoprotein with immobilized triazine dyes☆

Abstract We studied the interaction of α2-HS-glycoprotein with immobilized Cibacron blue F3-GA (Blue A) and Procion red HE-3B (Red A). When whole plasma was applied on the Blue A, α2-HS-glycoprotein remained unbound, together with other plasma proteins. In contrast, when this fraction was applied on the Red A, α2-HS-glycoprotein was shown to bind tightly and was eluted with a linear sodium chloride gradient between 0.5 and 0.8 M. This proved to be a useful two-step technique for the purification of α2-HS-glycoprotein. Further characterization revealed that the protein appeared homogeneous by immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis with yields greater than 30%. A small (

Altered configuration of Gc on the plasma membrane of transformed and malignant human B lymphocytes

Normal human peripheral blood B cells exhibit strong membrane fluorescence for Gc (vitamin D-binding protein), and this protein can form a close spatial relationship with integral membrane immunoglobulin (mIg) with evidence of codistribution in the lipid bilayer. In contrast, fluorescence for both Gc and mIg has been found in this study to be weak or absent in several B lymphoblastoid cell lines and in chronic lymphocytic leukemia B cells. Moreover, the comobility of these components, where detectable, was also impaired. In abnormal B cells, the intensity of membrane fluorescence for Gc was substantially increased after crosslinking of mIg with antibody, and the latter was also associated with increased specific radioiodination of Gc by lactoperioxidase. These results indicate that Gc can apparently become displaced under certain circumstances within or through the lipid bilayer. The altered content or membrane topography of Gc in such abnormal B cells might be associated with impaired expression and mobility of mIg.

High levels of group-specific component (vitamin-d-binding protein) in the cerebrospinal fluid of infants aged <2 months

Because of the possible involvement of group-specific component (Gc) or vitamin-D-binding protein in the immunological functions of mononuclear cells and the increased risk of central nervous system i