Human serum alpha 2HS-glycoprotein modulates in vitro bone resorption.

We previously isolated a family of bone-resorbing proteins from human cancer ascites fluid and established that the three purified bone-resorbing proteins were chemically and immunochemically related to each other and to alpha-2HS glycoprotein (alpha 2HS). After this finding we purified the normal human serum counterpart of these ascites proteins and studied its effects on bone resorption. The bone-resorbing properties of normal human serum alpha 2HS were examined in vitro over a wide dose range. This normal human serum glycoprotein had a biphasic effect on 45Ca2+ release from bone. More specifically, this protein stimulated bone resorption at the lower concentrations tested, with a maximum effect [treated over control ratio of 2.5 +/- 0.30 (+/- SE); P less than 0.01] at 40 micrograms/mL. In contrast, at doses above 40 micrograms/mL, a sharp decline in calcium mobilization occurred, with a return to baseline occurring above 80 micrograms/mL. These results suggest that serum alpha 2HS may participate in the regulation of bone metabolism in vivo.

Three forms of BRP-2 (bone resorptive proteins) from human cancer ascites fluid and their relationship to human serum alpha-2 HS-glycoprotein.

Two new forms of BRP-2, a previously described bone resorptive protein, were purified from ascites fluids obtained from patients with hypercalcemia and metastatic bone cancer. The apparent molecular weights of BRP-2 and of these two proteins were 52,000, 48,000, and 46,000, respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The three proteins have essentially the same amino acid compositions but differ with respect to their carbohydrate moieties. The amino-terminal amino acid sequences of the three glycoproteins were identical to each other as well as to human serum alpha 2HS-(human serum) glycoprotein. The relationship of the three forms of BRP-2 to alpha 2HS was also established immunochemically. The ascites proteins, as well as alpha 2HS, on a molar basis, were approximately one-tenth as potent as bovine parathyroid hormone fragment (1-34) in their abilities to stimulate calcium release from bone in vitro. This study describes for the first time a possible function for human serum alpha 2HS.

Synthetic human parathyroid hormone fragment stimulates prostaglandin E2 synthesis by chick calvariae.

Synthetic human PTH N-terminal 1-34 peptide [hPTH-(1-34)] stimulates prostaglandin (PG) production by chick calvariae in culture. PGE2 was the predominant PG found by both RIA and HPLC. Stimulation of PGE2 synthesis was significant at 50 ng/ml (1.2 X 10(-8) M) hPTH-(1-34) and was dose dependent at concentrations up to 0.6 microgram/ml (1.4 X 10(-7) M). Continuous exposure of calvariae to hPTH-(1-34) showed that PGE2 production increased significantly by 1 h, reached a maximum at 24 h, and then persisted over 96 h. Indomethacin at concentrations above 5 X 10(-7) M inhibited PGE2 synthesis by both control and hPTH-(1-34)-treated bones. PTH-(1-34) stimulated bone cells to convert arachidonic acid to PGE2, but did not activate the bone to release stored arachidonate. In summary, our results show that hPTH-(1-34) stimulates PGE2 synthesis by chick calvariae. This endogenous PGE2 may be involved in bone remodeling.

Purification and partial characterization of a Mr 52,000 glycoprotein from human cancer ascites fluid which stimulates bone resorption in vitro.

A glycoprotein which stimulates bone resorption in vitro has been purified at least 375-fold from human cancer ascites fluid. Purification was accomplished utilizing fractionation with ammonium sulfate, anion exchange chromatography, high-performance size exclusion chromatography, adsorptive chromatography on hydroxylapatite, and high-performance anion exchange chromatography. The homogeneous bone-resorptive protein possesses an apparent molecular weight of 52,000 as calculated from electrophoresis in sodium dodecyl sulfate:polyacrylamide gels. On polyacrylamide gels at pH 8.9 in the absence of sodium dodecyl sulfate, the bone-resorptive protein migrates with the mobility of an alpha 2-globulin. The amino acid composition of this protein is marked by the absence of methionine and by a relatively high content of nonpolar amino acids. The protein possesses a single amino- and a single carboxyl-terminal amino acid, alanine, and leucine, respectively. The carbohydrate moiety, comprising 20% of the total weight, consists of neutral hexoses, galactosamine and glucosamine, and sialic acids. With respect to its biological activity, the ascites glycoprotein is one-fourth as potent as parathyroid hormone, on a molar basis, in its ability to stimulate calcium release from bone explants in vitro. Calcium release is significantly decreased on heating at 60 degrees C but is not inhibited by indomethacin.

Purification and partial characterization of a protein from cancer ascites fluid which stimulates the resorption of bone explants in vitro.

A protein capable of stimulating bone resorption in vitro has been purified approximately 1250-fold from cancer ascites fluid. Purification was accomplished employing successive fractionation with ammonium sulfate, ion exchange, and Cibacron blue affinity chromatography, isoelectric focusing, and selective adsorption on hydroxylapatite. The bone-resorptive protein obtained by this procedure appeared homogeneous in polyacrylamide gels at pH 9.5, migrating with the mobility of an alpha 2-globulin, and in sodium dodecyl sulfate polyacrylamide gels from which an apparent molecular weight of 43,000 was calculated. The amino acid composition of the bone-resorptive protein distinguished itself by the absence of methionine and by its relatively high content of glycine (17%) and proline (11%). Furthermore, the protein possesses a single NH2-terminal amino acid residue (glycine). The ascites protein was found to contain 19% carbohydrate by weight including a high content of sialic acid (15 residues/mol) as compared to the other sugars (27 residues/mol). As to its biological properties, the homogeneous ascites glycoprotein proved to be as potent as parathyroid hormone in its ability to stimulate bone resorption in vitro.

The microheterogeneity of human plasma alpha1-acid glycoprotein.

alpha1-Acid glycoprotein was isolated in the homogeneous state from the plasma of 33 normal individuals and subjected to analytical isoelectric focusing before and after treatment with neuraminidase. The native glycoprotein preparations, resolved into 6 to 8 bands, were quantitated and grouped into two classes according to the patterns obtained: One class exhibited a relatively anodic and the other a relatively cathodic distribution of the protein bands. The isoelectric points of these bands ranged from pH 2.90 to 3.30. After treatment with neuraminidase the resulting asialo-glycoproteins were also quantitated and afforded two types of fundamentally different patterns from those mentioned above, namely one type with one and the other type with two main bands and both exhibiting several minor components. The isoelectric points of the main bands were found to be of pH 4.55 and 4.70 while those of the minor bands were at both the anodic and cathodic side of the major bands. No apparent relationship between the patterns of the native and those of the asialo-glycoproteins could be established. In addition, a new variant was noted whose major band focused at a pH of 5.0. The microheterogeneity of alpha1-acid glycoprotein is thus interpreted to be due to the amino acid replacements of this protein in combination with the linkages of the sialyl to the galactosyl residues in the native protein.

Effects of a bone resorptive factor from human cancer ascites fluid on rat bone cell calcium and cyclic AMP.

The effects of a bone resorptive protein isolated from human cancer ascites fluid on bone cell calcium and cyclic AMP were studied with fetal rat cells. The osteoclast-activating factor increased bone cell calcium uptake at 37 degrees C and 4 degrees C with no direct effects on calcium efflux. Concentrations of the resorptive factor that increased in vitro bone resorption and cell calcium uptake had no effect on cyclic AMP. The effects of the protein on calcium uptake were not specific for bone cells, and large increases were also observed in isolated fetal rat skin cells. These studies suggest that increases in the permeability of the cell membrane to calcium are involved in the mechanism of action of the ascites fluid resorptive protein.

Isolation of a bone-resorptive factor from human cancer ascites fluid.

A protein fraction that induces the resorption of bone explants in organ culture was isolated from the ascitic fluid of patients with advanced cancer metastatic to the peritoneal cavity. Partial purification was achieved by means of gel filtration, affinity chromatography, and ion-exchange chromatography. The isolated fraction, the components of which have an apparent molecular weight of 60,000, was found to be heterogeneous by disc gel electrophoresis and to be composed primarily of proteins with relatively acidic electrophoretic properties. The specific bone-resorptive activity of this protein fraction was greatly increased over that of the unfractionated starting material, and the activity could be completely destroyed upon incubation with pronase and on heating. As determined by immunoassay and extraction procedures with various solvents, the bone-resorptive action of the isolated fraction was not attributable to the presence of parathyroid hormone, prostaglandin E2 or vitamin D-like sterols. In parallel experiments the supernatants of phytohemagglutinin-stimulated normal human peripheral leukocytes were subjected to identical chromatographic techniques, and a proten fraction with a molecular weight of 60,000, which resembled the resorptive fraction isolated from cancer ascites fluid and which contained significant bone-resorptive activity, was also partially purified.

Sequence comparison of human plasma alpha1-acid glycoprotein and the immunoglobulins.

The amino acid sequence of human plasma alpha1-acid glycoprotein, upon comparison with the sequences of other blood proteins, was shown to possess significant similarity with the immunoglobulins. Employing direct and corrected sequence identity, the average mutation value and two different computer comparisons for the evaluation of sequence similarity, the following two regions of this alpha-globulin, which account for approximately half of the total amino acid sequence of the protein, were found to possess sequence similarity with the immunoglobulins. a) The region from residues 77 through 125 proved to be related to the variable region of several human H and L chains, and b) the region from residues 136 through 166 was found to be related not only to the constant region of a human and a mouse L chain but also to the third and fourth constant region of a rabbit and a human H chain, respectively. These results suggest that alpha1-acid glycoprotein is probably related to the immunoglobulins and further suggest that it possibly diverged from the immunoglobulin evolutionary tree prior to the formation of the primitive L chain.

A low molecular weight glycosaminoglycan from the human aorta.

A low mol. wt, dialyzable glycosaminoglycan was isolated from human aorta and was found to be homogeneous on 2 dimensional electrophoresis. As judged by its electrophoretic mobilities and its hydrolysis by chondroitin sulfatase ABC, it was concluded that this hitherto unknown glycosaminoglycan is an oversulfated chondroitin sulfate.

Association of a circulating immunosuppressive polypeptide with operative and accidental trauma.

The serum from 109 traumatized patients was examined for immunosuppressive activity which might explain diminished host immune responsiveness following operative or accidental injury. Twenty-eight fo 31 (90%) severely tralmatized patients, 25 of 60 (42%) moderately traumatized patients, and 0 of 18 minimally traumatized patients developed serum which suppressed the response of normal human lymphocytes to phytohemagglutinin. The degree and duration of serum immunosuppressive activity paralleled the severity of the clinical course but did not correlate with serum cortisol or barbiturate levels. Suppressive sera were not cytotoxic. The immunosuppressive factor(s) was contained in a low molecular weight (less than 10,000 daltons) peptide fraction and was present in 5--10 times the amount recoverable from normal serum. By size and activity the trauma serum factor resembled immunoregulatory alpha globulin, a naturally-occurring serum inhibitor of T-lymphocyte reactions. Thus, depressed immunoreactivity following trauma may be due in part to high concentrations of an endogenous immunosuppressive polypeptide.

Isolation of an immunosuppressive peptide fraction from the serum of cancer patients.

An immunosuppressive peptide fraction was isolated by means of gel filtration, membrane partition, and ion-exchange chromatography from the sera of patients hospitalized for cancer. The resulting peptide fraction, which was heterogeneous as judged by high-voltage electrophoresis, was found to suppress both phytohemagglutinin-induced proliferation of lymphocytes in vitro and the in vivo induction of splenic plaque-forming cells in mice. The specific activity of the peptide fraction, which was isolated from the sera of cancer patients, was significantly increased over that of the unfractionated starting material. Moreover, in control experiments, when the sera of normals or non-cancer-bearing hospitalized individuals were subjected to the same chromatographic techniques, no active peptide fraction could be obtained.