Interaction among hepatocyte-stimulating factors, interleukin 1, and glucocorticoids for regulation of acute phase plasma proteins in human hepatoma (HepG2) cells

Human hepatoma (HepG2) cells respond to unfractionated conditioned media of human squamous carcinoma (COLO-16) cells and lipopolysaccharide-stimulated human peripheral blood monocytes by increasing the synthesis of alpha 1-acid glycoprotein, haptoglobin, complement C3, alpha 1-antichymotrypsin, alpha 1-antitrypsin, and fibrinogen, while decreasing the synthesis of albumin. The regulation of the acute phase proteins is mediated by hepatocyte-stimulating factors (HSF) and interleukin 1 (IL-1) present in the conditioned medium. Purified HSF-I from COLO-16 cells stimulates preferentially alpha 1-acid glycoprotein synthesis, whereas COLO-HSF-II stimulates preferentially the synthesis of haptoglobin, fibrinogen, and alpha 1-antitrypsin. HSF from monocytes, which has been identified as interferon-beta 2 (B cell stimulating factor-2), displayed the same activity as COLO-HSF-II. Dexamethasone alone had no effect on acute phase plasma protein synthesis but enhanced the response to various HSF severalfold. IL-1 had a relatively low stimulatory activity on the synthesis of alpha 1-acid glycoprotein, haptoglobin, and alpha 1-antichymotrypsin but strongly reduced the basal expression of fibrinogen. The only synergistic action between IL-1 and HSF (or interferon-beta 2) was noted for the synthesis of alpha 1-acid glycoprotein. Tumor necrosis factor active on other hepatic cells failed to modulate significantly the expression of any plasma proteins in HepG2 cells. These studies showed that for an optimal HepG2-cell response a combination of HSF (or interferon-beta 2), IL-1, and dexamethasone is needed. This finding might indicate the identity of some of those hormones involved in regulation of the hepatic acute phase response in vivo.     

The permeability of EUDRAGIT RL and HEMA-MMA microcapsules to glucose and inulin

Measurement of the rate of glucose diffusion from EUDGRAGIT RL and HEMA-MMA microcapsules coupled with a Thiele modulus/Biot number analysis of the glucose utilization rate suggests that pancreatic islets and CHO (Chinese hamster ovary) cells (at moderate to high cell densities) should not be adversely affected by the diffusion restrictions associated with these capsule membranes. The mass transfer coefficients for glucose at 20 degrees C were of the same order of magnitude for both capsules, based on release measurements: approximately 5 x 10(-6) cm/s for EUDRAGIT RL and approximately 2 x 10(-6) for HEMA-MMA. Inulin release from EUDRAGIT RL was slower than for glucose (mass transfer coefficient 14 +/- 4 x 10(-8) cm/s). The Thiele moduli were much less than 1, either for a single islet at the center of a capsule or CHO cells uniformly distributed throughout a capsule at 10(-6) cells/ mL, so that diffusion restrictions within the cells in EUDRAGIT RL or 800 mum HEMA-MMA capsules should be negligible. The ratio of external to internal diffusion resistance (Biot number) was less than 1, so that at most, only a small diffusion effect on glucose utilization should be expected (i.e., the overall effectiveness factors were greater than 0.8). These calculations were consistent with experimental observation of encapsulated islet behavior but not fully with CHO cell behavior. Permeability restricted cell viability and growth is potentially a major limitation of encapsulated cells; further analysis is warranted.     

Protein glycosylation regulates the externalization of two distinct classes of glucocorticoid-induced glycoproteins in rat hepatoma cells

The relationship of protein glycosylation to the externalization of glucocorticoid inducible alpha1-acid glycoprotein and mouse mammary tumor virus glycoproteins was examined in M1.54, a clonal population of mouse mammary tumor virus-infected rat hepatoma cells. Multiple freeze-thaw of isolated microsomes revealed that while alpha 1-acid glycoprotein is carried through the cell as a soluble component of vesicles, extracellular viral glycoproteins are initially membrane-associated. At concentrations of tunicamycin that specifically inhibited N-linked protein glycosylation, alpha 1-acid glycoprotein fractionated between the cellular and extracellular compartments. Thus, approximately one half of the newly synthesized, nonglycosylated (22,000 Mr) alpha 1-acid glycoprotein was rapidly secreted with kinetics similar to its glycosylated counterpart (release half-time of 60 min), while the remaining species first localized in an undefined intracellular compartment prior to its slow secretion (release half-time of 24 h). The same distribution of nonglycosylated alpha 1-acid glycoprotein was observed at various absolute levels of polypeptide, suggesting that this was not due simply to the saturation of an efficient secretory pathway at high polypeptide levels. In contrast to alpha 1-acid glycoprotein, no labeled viral antigens were released by tunicamycin-treated M1.54, while a nonglycosylated viral precursor glycopolyprotein was expressed intracellularly. Taken together, these results suggest that carbohydrate attachment strongly regulates the externalization of both alpha 1-acid glycoprotein and mouse mammary tumor virus species, which represent two distinct classes of extracellular glycoproteins.     

Metabolic activity of CHO fibroblasts in HEMA-MMA microcapsules

Chinese hamster ovary (CHO) fibroblast cells were microencapsulated in polyacrylate membranes (HEMA-MMA: 75% HEMA) via an interfacial precipitation process. The CHO cells were observed to grow in large aggregates, attached to each other instead of to the capsule wall. When CHO cells were encapsulated at high density (4 x 10(6) cells/mL), the initial metabolic activity in microcapsules, as determined by the MTT assay, correlated with the polymer-cell extrusion ratio, presumably because of the dependence of encapsulation efficiency on the relative flow rates. However, there was a large variation in the metabolic activity among individual microcapsules throughout the present study. Capsules with low encapsulation efficiency (at a "seeding" density of 4 x 10(6) cells/mL) exhibited a rapid increase in the metabolic activity during the following week. When CHO cells were encapsulated at low density (4 x 10(5) cells/mL), there was only a small increase in the metabolic activity. Only a small fraction ( approximately 5%) of the capsules exhibited a high level of metabolic activity and 40% of the capsules exhibited undetectable metabolic activity even after 2 weeks. We conclude that CHO cells, which served as model cells, survive the encapsulation process and retain an active metabolic state once enclosed by the HEMA-MMA membranes. However, the resultant microcapsules are extremely heterogeneous in the amount of retained metabolic activity.     

Secretion of high-mannose-type alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein by primary cultures of rat hepatocytes in the presence of the mannosidase I inhibitor 1-deoxymannojirimycin

Two different forms of alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein were found in primary cultures of rat hepatocytes. After a 2.5-h labeling period with [35S]methionine the high-mannose-type precursor of alpha 1-proteinase inhibitor (Mr 49000) and alpha 1-acid glycoprotein (Mr 39 000) and the mature-complex-type alpha 1-proteinase inhibitor (Mr 54 000) and alpha 1-acid glycoprotein (Mr 43 000-60 000) could be immunoprecipitated from the cells, but only the complex-type forms of the two glycoproteins were secreted into the hepatocyte media. When hepatocytes were incubated with the mannosidase I inhibitor 1-deoxymannojirimycin at a concentration of 4 mM, the 49 000-Mr form of alpha 1-proteinase inhibitor and the 39 000-Mr form of alpha 1-acid glycoprotein could be detected in the cells as well as in their media. Neither the secretion of alpha 1-proteinase inhibitor nor that of alpha 1-acid glycoprotein was impaired by 1-deoxymannojirimycin. While alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein, secreted by control cells, were resistant to endoglucosaminidase H, alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein, secreted by hepatocytes treated with 4 mM 1-deoxymannojirimycin, could be deglycosylated by endoglucosaminidase H. When the [3H]mannose-labeled oligosaccharides of alpha 1-proteinase inhibitor, secreted by 1-deoxymannojirimycin-treated hepatocytes, were cleaved off by endoglucosaminidase H and analyzed by Bio-Gel P-4 chromatography, they eluted at the position of Man9GlcNAc, indicating that mannosidase I had been efficiently inhibited. 1-Deoxymannojirimycin did not inhibit the synthesis or the cotranslational N-glycosylation of alpha 1-proteinase inhibitor or alpha 1-acid glycoprotein.     

Effect of the threonine analog beta-hydroxynorvaline on the glycosylation and secretion of alpha 1-acid glycoprotein by rat hepatocytes

The threonine analog beta-hydroxynorvaline (Hnv) is an inhibitor of asparagine-linked glycosylation. In the presence of the analog hepatocytes synthesized immunoreactive alpha 1-acid glycoprotein with 0-6 oligosaccharide chains. Pulse-chase experiments were conducted to compare the rates of secretion of alpha 1-acid glycoprotein from untreated, tunicamycin-treated, and Hnv-treated cells. Partially glycosylated (1-5 oligosaccharide chains) and unglycosylated (tunicamycin-inhibited) molecules exited the cells more slowly than native alpha 1-acid glycoprotein. In addition, secretion of fully glycosylated (6 oligosaccharide chains) alpha 1-acid glycoprotein was retarded in Hnv-treated cells when compared to controls. The slowest rate of secretion was exhibited by the unglycosylated form from Hnv-treated cells. These results suggest that Hnv-induced changes either in the extent of glycosylation or in the peptide sequence of alpha 1-acid glycoprotein can interfere with its transport through the cell. The major intracellular forms of alpha 1-acid glycoprotein from control and Hnv-treated cells were endoglycosidase H-sensitive and contained Man9-8 GlcNAc2 oligosaccharide structures. The oligosaccharide chains on the secreted molecules from control and Hnv-treated cells were entirely of the endoglycosidase H-resistant, complex type.     

Synthesis and secretion of some plasma proteins by tissue slices of Morris hepatoma 7777 and liver from control and turpentine-injected rats

Slices of Morris hepatoma 7777 or rat liver isolated from control or turpentine-injected rats were incubated for 2 h with 14C-leucine. Radioactivities incorporated into albumin, alpha-fetoprotein, fibrinogen, alpha 1-AP-globulin, haptoglobin and alpha 1-acid glycoprotein were determined after the proteins had been isolated from the incubation medium or tissue homogenate by immunoprecipitation with monospecific antisera. It was found that hepatoma synthesizes fibrinogen, alpha 1-AP-globulin and alpha 1-acid glycoprotein in the amounts comparable to rat liver, whereas formation of albumin and haptoglobin is reduced 5- to 10-fold. Local inflammation elicited by injection of turpentine to tissue donors increased formation of acute-phase protein in liver slices but had no effect on synthesis of these proteins in preparations of Morris hepatoma, although certain ultrastructural changes in the Golgi complex were observed not only in the liver but also in the tumour.     

Recombinant human interleukin-6 (IL-6/BSF-2/HSF) regulates the synthesis of acute phase proteins in human hepatocytes

Recombinant human IL-6 (rhIL-6) is a potent inducer of the synthesis of acute phase proteins in adult human hepatocytes. A wide spectrum of acute phase proteins is regulated by this mediator. After labeling of rhIL-6 stimulated human hepatocytes with [35S]methionine acute phase protein synthesis was measured by immunoprecipitation. Serum amyloid A, C-reactive protein, haptoglobin, alpha 1-antichymotrypsin and fibrinogen were strongly induced (26-, 23-, 8.6-, 4.6- and 3.8-fold increases, respectively). Moderate increases were found for alpha 1-antitrypsin (2.7-fold) and alpha 1-acid glycoprotein (2.7-fold). RhIL-6 had no effect on alpha 2-macroglobulin, whereas fibronectin, albumin and transferrin decreased to 64, 56 and 55% of controls. In the cases of serum amyloid A, haptoglobin, alpha 1-antichymotrypsin, alpha 1-antitrypsin and alpha 1-acid glycoprotein, dexamethasone enhanced the action of rhIL-6. We conclude that rhIL-6 controls the acute phase response in human liver cells.     

Distinct sets of acute phase plasma proteins are stimulated by separate human hepatocyte-stimulating factors and monokines in rat hepatoma cells

A subline of the rat hepatoma (H-35) cells has been identified which responds to hepatocyte-stimulating factors (HSFs) of human squamous carcinoma cells by increased synthesis of all major rat acute phase plasma proteins. The regulation occurs at the level of mRNA. Two HSFs (HSF-I and HSF-II) have been purified from conditioned medium of the squamous carcinoma cells. HSF-I is a protein with an Mr = 18,000 and pI 5.5, and HSF-II is a glycoprotein with an Mr = 34,000 and a broad, neutral to basic charge. In H-35 cells, HSF-I predominantly stimulates the synthesis of complement C3 and haptoglobin and acts synergistically with dexamethasone to stimulate alpha 1-acid glycoprotein. HSF-II stimulates cysteine protease inhibitor, alpha 1-antichymotrypsin, alpha 1-antitrypsin, fibrinogen, and hemopexin, and acts synergistically with dexamethasone to stimulate alpha 2-macroglobulin. Each HSF is between 10 and 100 times less effective in regulating proteins of the other set. Human tumor necrosis factor and interleukin-1 increase complement C3, haptoglobin, and alpha 1-acid glycoprotein, as does HSF-I, but are unable to modulate any of the other acute phase proteins. The monokines differ from HSF-I is their low activity in HepG2 cells and rat hepatocytes.     

Induction of alpha 1-acid glycoprotein by recombinant human interleukin-1 in rat hepatoma cells

The induction of alpha 1-acid glycoprotein mRNA by recombinant murine interleukin-1, recombinant human interleukin-1 alpha, and recombinant human interleukin-1 beta has been studied in the rat hepatoma cell line Fao. Whereas the stimulatory capacities of recombinant human interleukin-1 alpha and recombinant murine interleukin-1 were almost identical, the concentrations of recombinant human interleukin-1 beta needed for half-maximal induction of alpha 1-acid glycoprotein mRNA were lower by three orders of magnitude. A 60-fold increase in alpha 1-acid glycoprotein mRNA levels was observed 18 h after the addition of recombinant interleukin-1 beta. In parallel albumin mRNA levels decreased to about 30%. The alpha 1-acid glycoprotein mRNA induction was strictly dependent on the presence of dexamethasone. For a full stimulation dexamethasone concentrations of greater than 10(-7) M were needed, whereas concentrations of less than 10(-12) M were ineffective. The increase in alpha 1-acid glycoprotein mRNA after recombinant human interleukin-1 beta was followed by a 36-fold stimulation in alpha 1-acid glycoprotein synthesis and secretion. When protein synthesis was blocked by either cycloheximide, puromycin, or emetine, the induction of alpha 1-acid glycoprotein mRNA by recombinant human interleukin-1 beta was impaired suggesting the involvement of a short-lived protein in the induction of alpha 1-acid glycoprotein mRNA.     

Acute phase protein alpha 1-acid glycoprotein interacts with plasminogen activator inhibitor type 1 and stabilizes its inhibitory activity.

alpha(1)-Acid glycoprotein, one of the major acute phase proteins, was found to interact with plasminogen activator inhibitor type 1 (PAI-1) and to stabilize its inhibitory activity toward plasminogen activators. This conclusion is based on the following observations: (a) alpha(1)-acid glycoprotein was identified to bind PAI-1 by a yeast two-hybrid system. Three of 10 positive clones identified by this method to interact with PAI-1 contained almost the entire sequence of alpha(1)-acid glycoprotein; (b) this protein formed complexes with PAI-1 that could be immunoprecipitated from both the incubation mixtures and blood plasma by specific antibodies to either PAI-1 or alpha(1)-acid glycoprotein. Such complexes could be also detected by a solid phase binding assay; and (c) the real-time bimolecular interactions monitored by surface plasmon resonance indicated that the complex of alpha(1)-acid glycoprotein with PAI-1 is less stable than that formed by vitronectin with PAI-1, but in both cases, the apparent K(D) values were in the range of strong interactions (4.51 + 1.33 and 0.58 + 0.07 nm, respectively). The on rate for binding of PAI-1 to alpha(1)-glycoprotein or vitronectin differed by 2-fold, indicating much faster complex formation by vitronectin than by alpha(1)-acid glycoprotein. On the other hand, dissociation of PAI-1 bound to vitronectin was much slower than that from the alpha(1)-acid glycoprotein, as indicated by 4-fold lower k(off) values. Furthermore, the PAI-1 activity toward urokinase-type plasminogen activator and tissue-type plasminogen activator was significantly prolonged in the presence of alpha(1)-acid glycoprotein. These observations suggest that the complex of PAI-1 with alpha(1)-acid glycoprotein can play a role as an alternative reservoir of the physiologically active form of the inhibitor, particularly during inflammation or other acute phase reactions.     

Insulin modulation of acute-phase protein production in a human hepatoma cell line.

Insulin is widely used as a growth factor in hepatocyte culture but its effect on the production of acute-phase proteins has not been studied. By measuring four positive (fibrinogen, alpha 1-antitrypsin, alpha 1-acid glycoprotein, and alpha 1-antichymotrypsin) and four negative (albumin, prealbumin, transferrin, and retinol binding protein) acute-phase proteins produced by the Hep G2 hepatoma cell line, we have shown that insulin is an important modulator of acute-phase protein production. Our data show that insulin is able to inhibit the synthesis of prealbumin, transferrin, and fibrinogen. The results also show a complex interaction between insulin, interleukin 6, and glucocorticoids because insulin is able to inhibit the dexamethasone induction of alpha 1-antichymotrypsin, and in the presence of interleukin 6, dexamethasone is able to regulate the production of fibrinogen and prealbumin. The regulatory role of insulin in fibrinogen production was confirmed by pulse chase labeling followed by immunoprecipitation and fluorography.     

Induction of several acute-phase protein genes by heavy metals: a new class of metal-responsive genes.

Acute-phase reactants, metallothioneins, and heat-shock proteins are the products of three families of genes that respond to glucocorticoids and cytokines. Metallothioneins and heat-shock proteins, however, are also stimulated by heavy metals, whereas very little is known about the effect of heavy metals on acute-phase-reactant genes. We have studied the effect of heavy metals (Hg, Cd, Pb, Cu, Ni, and Zn) and Mg on the acute-phase reactants alpha 1-acid glycoprotein, C-reactive protein, alpha 1-antitrypsin and alpha 1-antichymotrypsin. alpha 1-Acid glycoprotein and C-reactive protein mRNA levels were increased severalfold in livers of heavy-metal-treated Balb/c mice. The strongest induction was mediated by Hg, followed in order of response by Cd greater than Pb greater than Cu greater than Ni greater than Zn greater than Mg. None of the metals affected the mRNA levels of albumin, alpha 1-antitrypsin, and alpha 1-antichymotrypsin. Furthermore, failure to repress albumin, a negative acute-phase reactant, indicated that the induction of these genes was not due to a metal-mediated inflammatory response. The metals also induced alpha 1-acid glycoprotein and C-reactive protein in adrenalectomized animals, indicating that induction by the heavy metals is not mediated by the glucocorticoid induction pathway. Sequence analysis has revealed a region of homology to metal-responsive elements in the alpha 1-acid glycoprotein and C-reactive protein promoters. Additionally, an alpha 1-acid glycoprotein expression vector, pAGP(-595)CAT, responded to Hg and Cd when transfected into human HepG2 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

A carcinoembryonic antigen (CEA) binding protein from ascites influences CEA uptake by macrophages

A variant of CEA which is less readily endocytosed by macrophages has been isolated from malignant ascites. In vivo, CEA is cleared more slowly by the liver (t1/2 = 15.1 minutes) than CEAs isolated from hepatic metastases (t1/2 = 3.1 minutes). In vitro, rat and human Kupffer cells and rat alveolar macrophages endocytose this CEA less effectively. This slow clearing form of CEA is associated with a smaller (45kD) acidic glycoprotein (CORA) with which it forms a stable complex. CORA can be visualized on reducing gels but not on non reducing gels or by HPLC run under non reducing conditions. This suggests a non-covalent complex between the two glycoproteins. Analysis of protein conformation by circular dichroism revealed changes in the ascites CEA consistent with binding of CORA to the molecule. Western blot showed that CORA crossreacts with antisera to alpha 1-acid glycoprotein and double immunodiffusion demonstrated cross-reactivity but not identify. Sequencing of CNBr peptides showed sequence homology with alpha 1-acid glycoprotein but areas of unique sequence were also found. It is suggested that binding of CORA to CEA blocks the macrophage receptor binding of CEA.     

Interaction between calcofluor white and carbohydrates of alpha 1-acid glycoprotein.

Interactions between the fluorescent probe, calcofluor white, and human serum albumin (HSA) and alpha 1-acid glycoprotein (orosomucoid) are compared. The two proteins have comparable isoelectric points, but alpha 1-acid glycoprotein is highly glycosylated (40% of glycans by weight), while the serum albumin is not. Binding of calcofluor to the proteins induces an increase in both the fluorescence anisotropy and the fluorescence intensity of the fluorophore. Also, we found that the calcofluor exhibits a fluorescence emission with a maximum located at 432, 415 or 445 nm, respectively, in the absence of proteins, in the presence of HSA, and in the presence of alpha 1-acid glycoprotein. The stoichiometries of the calcofluor-serum albumin and calcofluor-alpha 1-acid glycoprotein complexes are 2:1 and 1:1, respectively. The association constants are 0.04 and 0.15 microM-1, respectively. The calcofluor does not interact with Lens culinaris agglutinin (LCA), although the protein has a hydrophobic site. Nevertheless, one cannot exclude that the binding of the fluorophore to the HSA is nonspecific. Our results, when compared with those obtained with calcofluor dissolved in the hydrophobic solvent isobutanol, and with the fluorescent probe, potassium 6-(p-toluidino)-2-naphthalenesulfonate (TNS), bound to alpha 1-acid glycoprotein, indicate that the emission of calcofluor bound to HSA occurs from a hydrophobic state, while that of calcofluor bound to alpha 1-acid glycoprotein occurs from a hydrophilic state. The fluorescence intensity of calcofluor decreases in the presence of carbohydrates isolated from alpha 1-acid glycoprotein, while it increases in the presence of alpha 1-cellulose. Thus, calcofluor interacts mainly with the glycan moiety of alpha 1-acid glycoprotein, and its fluorescence is sensitive to the secondary structure of the glycans.     

Interaction between calcofluor white and carbohydrates of alpha 1-acid glycoprotein.

Interactions between the fluorescent probe, calcofluor white, and human serum albumin (HSA) and alpha 1-acid glycoprotein (orosomucoid) are compared. The two proteins have comparable isoelectric points, but alpha 1-acid glycoprotein is highly glycosylated (40% of glycans by weight), while the serum albumin is not. Binding of calcofluor to the proteins induces an increase in both the fluorescence anisotropy and the fluorescence intensity of the fluorophore. Also, we found that the calcofluor exhibits a fluorescence emission with a maximum located at 432, 415 or 445 nm, respectively, in the absence of proteins, in the presence of HSA, and in the presence of alpha 1-acid glycoprotein. The stoichiometries of the calcofluor-serum albumin and calcofluor-alpha 1-acid glycoprotein complexes are 2:1 and 1:1, respectively. The association constants are 0.04 and 0.15 microM-1, respectively. The calcofluor does not interact with Lens culinaris agglutinin (LCA), although the protein has a hydrophobic site. Nevertheless, one cannot exclude that the binding of the fluorophore to the HSA is nonspecific. Our results, when compared with those obtained with calcofluor dissolved in the hydrophobic solvent isobutanol, and with the fluorescent probe, potassium 6-(p-toluidino)-2-naphthalenesulfonate (TNS), bound to alpha 1-acid glycoprotein, indicate that the emission of calcofluor bound to HSA occurs from a hydrophobic state, while that of calcofluor bound to alpha 1-acid glycoprotein occurs from a hydrophilic state. The fluorescence intensity of calcofluor decreases in the presence of carbohydrates isolated from alpha 1-acid glycoprotein, while it increases in the presence of alpha 1-cellulose. Thus, calcofluor interacts mainly with the glycan moiety of alpha 1-acid glycoprotein, and its fluorescence is sensitive to the secondary structure of the glycans.     

The association of acute phase protein genes with the nuclear matrix of rat liver during experimental inflammation

The association of acute phase protein genes with the nuclear matrix in livers from healthy rats and rats suffering from inflammation was studied. alpha 1-Acid glycoprotein and transthyretin are synthesized at low levels in normal liver and no matrix association of their genes was observed. Albumin, transferrin and the beta-chain of fibrinogen are synthesized at much higher levels in normal liver and their genes were found to be associated with the nuclear matrix. An inflammation induced increase in synthesis of alpha 1-acid glycoprotein and the beta-chain of fibrinogen resulted in stronger matrix association of their genes. However, inflammation induced decrease in the synthesis of albumin did not influence matrix association of its gene.     

Hierarchical assembly and the onset of banding in fibrous long spacing collagen revealed by atomic force microscopy.

The mechanism of formation of fibrillar collagen with a banding periodicity much greater than the 67 nm of native collagen, i.e. the so-called fibrous long spacing (FLS) collagen, has been speculated upon, but has not been previously studied experimentally from a detailed structural perspective. In vitro, such fibrils, with banding periodicity of approximately 270 nm, may be produced by dialysis of an acidic solution of type I collagen and alpha(1)-acid glycoprotein against deionized water. FLS collagen assembly was investigated by visualization of assembly intermediates that were formed during the course of dialysis using atomic force microscopy. Below pH 4, thin, curly nonbanded fibrils were formed. When the dialysis solution reached approximately pH 4, thin, filamentous structures that showed protrusions spaced at approximately 270 nm were seen. As the pH increased, these protofibrils appeared to associate loosely into larger fibrils with clear approximately 270 nm banding which increased in diameter and compactness, such that by approximately pH 4.6, mature FLS collagen fibrils begin to be observed with increasing frequency. These results suggest that there are aspects of a stepwise process in the formation of FLS collagen, and that the banding pattern arises quite early and very specifically in this process. It is proposed that typical 4D-period staggered microfibril subunits assemble laterally with minimal stagger between adjacent fibrils. alpha(1)-Acid glycoprotein presumably promotes this otherwise abnormal lateral assembly over native-type self-assembly. Cocoon-like fibrils, which are hundreds of nanometers in diameter and 10-20 microm in length, were found to coexist with mature FLS fibrils.     

Steroid-protein interactions. XXXIV. Chemical modification of alpha1-acid glycoprotein for characterization of the progesterone binding site.

The nature of the steroid binding site in alpha1-acid glycoprotein (orosomucoid) was investigated by chemical modification of individual amino acids and subsequent examination of the binding affinity for progesterone. Equilibrium dialyses were performed under conditions that excluded contact with human skin. Reaction of the lysyl residues with trinitrobenzenesulfonic acid or arylisocyanates resulted in a reduction of active sites. In an alternate approach, one lysyl residue of alpha1-acid glycoprotein was protected from modification by trinitrobenzenesulfonic acid when progesterone was present to form the complex with alpha1-acid glycoprotein. We conclude that a lysyl residue is located in the binding site. Reaction of tetranitromethane with the tyrosine groups in alpha1-acid glycoprotein also reduced the number of active binding sites for progesterone. Again, a partial protection of this modification was seen in the presence of progesterone and other delta4-3-ketosteroids. The progesterone binding activity observed in the tyrosine-modified alpha1-acid glycoprotein by equilibrium dialysis and by fluorescence quenching titration can be interpreted best by the presence of one tyrosyl residue in the binding site, and involvement of a second tyrosine nearby. Modification of tryptophan in alpha1-acid glycoprotein by mild acid hydrolysis, N-bromosuccinimide, hydroxynitrobenzylbromide, and formic acid resulted in a decreased steroid binding; the formylation reaction was fully reversible. The approximate distance between progesterone and the tryptophan involved in the binding was calculated to be between 9.1 A and 14.1 A. When alpah1-acid glycoprotein was cleaved by the cyanogen bromide procedure according to Ikenaka et al. (1972, Biochemistry 11, 3817-3829), both the amino and the carboxyl fragment had a weak progesterone binding affinity which could be measured in 4 M NaCl. This result thus failed to specify the location of the steroid binding site in alpha1-acid glycoprotein. However, the closeness of tryptophan, lysine and tyrosine in the primary and presumably the tertiary structure of alpha1-acid glycoprotein is in agreement with the properties of the binding site suggested by our studies.