Cloning and nucleotide sequence of a human Zn-alpha 2-glycoprotein cDNA and chromosomal assignment of its gene

A cDNA clone of Zn-alpha 2-glycoprotein (Zn alpha 2gp) was isolated from a human prostate library. The amino acid sequence of prostate Zn alpha 2gp deduced from the nucleotide sequence was identical to the one previously reported on the Zn alpha 2gp protein purified from human blood plasma, except at three positions: the 65th and 222nd amino acid residues were Gln (----Glu) and Glu (----Gln), and there was a two amino acid insertion (Ile-Phe) between the 75th (Glu) and 76th (Met) amino acids. Southern blot analysis of human genomic DNA, however, suggested a single gene encoding Zn alpha 2gp. Using a panel of rodent-human somatic cell hybrids, the Zn alpha 2 gp gene was assigned to human chromosome 7.     

The 39-kDa receptor-associated protein interacts with two members of the low density lipoprotein receptor family, alpha 2-macroglobulin receptor and glycoprotein 330.

The 39-kDa receptor-associated protein (RAP) binds to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) and inhibits binding of ligands to this receptor. The in vivo function of RAP may be to regulate ligand binding and/or assist in the correct biosynthetic processing or trafficking of the alpha 2MR/LRP. Here we show that RAP binds another putative receptor, the kidney glycoprotein 330 (gp330). Gp330 is a high molecular weight glycoprotein that is structurally similar to both the alpha 2MR/LRP and low density lipoprotein receptor. The ability of RAP to bind to gp330 was demonstrated by ligand blotting and solid phase binding assays, which showed that RAP binds to gp330 with high affinity (Kd = 8 nM). Exploiting the interaction of gp330 and RAP, we purified gp330 by affinity chromatography with a column of RAP coupled to Sepharose. Gp330 preparations obtained by this procedure were notably more homogeneous than those obtained by conventional methods. Immunocytochemical staining of human kidney sections localized RAP to the brush-border epithelium of proximal tubules. The fact that gp330 is also primarily expressed by proximal tubule epithelial cells strengthens the likelihood that the interaction between gp330 and RAP occurs in vivo. The functional significance of RAP binding to gp330 may be to antagonize ligand binding as has been demonstrated for the alpha 2MR/LRP or to assist in the biosynthetic processing and/or trafficking of this receptor.     

Fetuin and alpha-2HS glycoprotein induce alkaline phosphatase in epiphyseal growth plate chondrocytes.

A previously described chondrocyte alkaline phosphatase induction factor (CAP-IF) for chicken epiphyseal growth plate chondrocytes has been purified to SDS-PAGE homogeneity from fetal bovine serum by ammonium sulfate precipitation and by dye-ligand affinity (Affi-Gel Blue and Reactive Green-19 agarose) and hydroxyapatite column chromatographies. As determined by immunoprecipitation of [35S]methionine-labeled cellular proteins after 3 day treatment, this highly purified CAP-IF increases the level of AP and certain other membrane proteins 2- to 3-fold over control values. The pure protein of apparent 64.5 kDa molecular weight has been identified as fetuin by N-terminal amino acid sequencing. This was confirmed by the finding that high alkaline phosphatase (AP)-inducing activity is present in fetuin prepared by the Spiro method. However, fetuins prepared by the Pedersen or Deutsch procedures are inactive. At least half of the CAP-IF activity of fetuin was irreversibly destroyed by treatment with EDTA and addition of Zn2+ did not reactivate the EDTA-treated fetuin. Ascorbate synergistically enhanced the effect of fetuin on chondrocyte AP activity by over 8-fold during 3 day exposure. Because of the very high homology between fetuin and the A-chain of alpha 2-HS glycoprotein, we also tested and found that alpha 2HS glycoproteins from human serum and bovine bone are both strong AP inducers. Our findings suggest that the AP-inducing activity resides in a labile, cystatin/Zn(2+)-binding domain common to these related serum glycoproteins. These proteins appear to play a role in enhancing AP expression in normal growth plate cartilage differentiation.     

Isolation and characterization of the receptor on human neutrophils that mediates cellular adherence

The receptor on human neutrophils (polymorphonuclear leukocytes or PMN) that mediates cellular adherence has been purified from the peripheral blood PMN obtained from an individual with chronic myelogenous leukemia (CML). This receptor consists of two noncovalently associated subunits, designated alpha M (Mac-1 alpha, CD11b) (Mr = 170,000) and beta (Mac-1 beta, CDw18) (Mr = 100,000), respectively, which are identical on normal and CML PMN. The subunits were purified by monoclonal antibody 60.1-Sepharose (anti-alpha M) affinity chromatography and separated in 5-nmol quantities by high pressure liquid chromatography on a TSK-4000 gel filtration column. Subunits were characterized by amino acid composition, NH2-terminal amino acid sequence, and carbohydrate content. The NH2-terminal sequence of the human PMN alpha M subunit contains regions of homology with the human platelet glycoprotein IIb alpha. We conclude that nanomole amounts of individual alpha M and beta subunits of the receptor on human PMN that mediates cellular adherence can be isolated and separated using CML PMN.     

Purification and characterization of a novel metalloendopeptidase from Streptococcus cremoris H61. A metalloendopeptidase that recognizes the size of its substrate

An endopeptidase (LEP-II), which has a unique substrate specificity, was purified to homogeneity by conventional chromatographic techniques from Streptococcus cremoris H61. The enzyme was a metalloendopeptidase since it was inhibited by EDTA and 1,10-phenanthroline; the metal-depleted enzyme could be fully reactivated by micromolar levels of Zn2+ and was not inhibited by specific inhibitors for serine or thiol protease. The molecular mass of the enzyme was estimated to be 80 kDa by Sephacryl S-300 gel filtration and high-performance liquid chromatography with a TSK-G3000SW column. The enzyme consisted of two identical subunits and the N-terminal sequence of LEP-II was determined up to the 19th residue. Although the enzyme had a broad substrate specificity it specifically hydrolyzed the peptide bonds involving the amino groups of hydrophobic amino acid residues. Various small polypeptides, such as alpha s1-CN(f1-23), alpha s1-CN(f91-100), oxidized insulin B chain, glucagon and some biologically active peptides were hydrolyzed. However, a variety of larger polypeptides or proteins, such as alpha s1-CN(f1-54), alpha s1-CN(f61-123), alpha s1-CN(f136-196), alpha s1-casein, beta-casein, and kappa-casein were not hydrolyzed. LEP-II recognized the size of its substrates, which were limited below a molecular mass of about 3.5 kDa.     

Purification and characterization of a novel growth factor from human breast cancer cells.

We have purified and characterized a novel 30-kDa glycoprotein (gp30) with TGF alpha-like properties secreted from the estrogen receptor negative breast cancer cell line MDA-MB-231. This factor was immunoprecipitated by an anti-TGF alpha polyclonal antibody and also had TGF alpha-like biological activity, as assayed by EGF radioreceptor assay and anchorage-independent assays. In addition, the novel growth factor stimulated phosphorylation of the EGF receptor and erbB-2 receptor. However, the novel growth factor, unlike EGF and TGF alpha, bound to heparin-Sepharose. Purification of gp30 was obtained to apparent homogeneity by heparin affinity chromatography and subsequent reversed-phase chromatography. Tunicamycin treatment in vivo or N-glycanase deglycosylation in vitro revealed a putative precursor of approximately 22 kDa molecular mass in contrast to the "normal" 16-kDa precursor species for TGF alpha. In vitro translation of total mRNA from MDA-MB-231 cells confirmed the size of the putative precursor. Biochemical characterization of gp30 was begun by V8 protease digestion of the deglycosylated polypeptide and the translated products. Peptide mapping of V8-digested, immunoprecipitated material suggests that the amino acid sequence of this unique protein is distinct from mature TGF alpha and not the result of a posttranslational modification of the precursor. We conclude that this TGF alpha-like (gp30) polypeptide is a novel growth factor with agonistic activity for both EGF and erbB-2 receptors.     

GTP-binding proteins in human platelet membranes serving as the specific substrate of islet-activating protein, pertussis toxin

Two GTP-binding proteins serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, were purified from human platelet membranes as heterotrimers with an alpha beta gamma-subunit structure. The alpha of the major IAP substrate had a molecular mass of 40 kDa and differed from that of Gi 1 or Go previously purified from brain membranes. The partial amino acid sequences of the 40 kDa alpha completely matched with the sequences which were deduced from the nucleotide sequences of the human Gi 2 alpha gene. On the other hand, the alpha of the minor IAP substrate purified from human platelets was about 41 kDa and cross-reacted with an antibody raised against alpha of brain Gi 1 (Gi 1 alpha). These results indicate that the major IAP substrate present in human platelet membranes is a product of the Gi 2 alpha gene.     

A fibrinolytic metalloprotease from the fruiting bodies of an edible mushroom, Armillariella mellea

A fibrinolytic metalloprotease has been purified from the fruiting bodies of the edible honey mushroom (Armillariella mellea). The enzyme has a molecular weight of 18538.1508, as measured by MALDI-TOF mass spectrometry and includes Zn2+ ion as found by ICP/MS. The N-terminal amino acid sequence, XXYNGXTXSRQTTLV, do not match any known protein or open reading frame. It hydrolyzes fibrinogen as well as fibrin, but does not show any proteolytic activity for other blood proteins such as thrombin, human albumin, bovine albumin, human IgG, hemoglobin, or urokinase. This protease hydrolyzes both A alpha and B beta subunits of human fibrinogen with equal efficiency. The enzyme activity was strongly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzyme is a metalloprotease. No inhibition was found with PMSF, E-64, pepstatin, and 2-mercaptoethanol. The activity of the purified enzyme was slightly increased by Mg2+, Zn2+, and Co2+, but the enzyme was totally inhibited by Hg2+. It has broad substrate specificity for synthetic peptides, and a pH optimum at 7, suggested that the purified enzyme was a neutral protease. It was thermally stable up to 60 degrees C and the maximum fibrinolytic activity was at 55 degrees C.     

Purification of human alpha 2-plasmin inhibitor using monoclonal antibody column chromatography

alpha 2-Plasmin inhibitor (alpha 2PI) was purified directly from human plasma by using a monoclonal antibody affinity column, which recognizes the reactive site of alpha 2PI. alpha 2PI was eluted from the column under mild conditions with 50% v/v ethyleneglycol containing 0.05% Tween 80 in phosphate-buffered saline, pH 7.4. The yield was around 50% and the specific activity of the purified protein was the same as that of the best product of preparation conventionally purified alpha 2PI.     

Purification by Strep-Tactin Affinity Chromatography of a Delete Envelope gp51 Protein of Bovine Leukaemia Virus Expressed in Sf21 Insect Cells

Bovine leukaemia virus (BLV) causes disease in cattle and it is related to human T lymphotrofic viruses HTLV-1 and HTLV-2. The objective of this study was to express and purify deleted and stable forms of the gp51 envelope glycoprotein of BLV using a baculovirus system. Two forms of the gp51 were synthesised: one comprised the gp51 N-terminal 174 amino acids and a single 6xHis tag (∆_175-268gp51-His) and the second form contained the same amino acid sequence and a C-terminal Strep-tag II in addition to the 6xHis tag (∆_175-268gp51-STH). The two proteins were expressed and purified by immobilized metal-affinity chromatography (IMAC) or by Strep-Tactin column. The Strep-Tactin technology was more efficient than IMAC method and achieved a high pure recombinant deleted gp51. In addition the ∆_175-268gp51-STH protein was further concentrated by IMAC. This purified antigen could be used for the isolation of immunoreactive molecules and to develop a competitive ELISA test.     

Purification of pancreatic phospholipase A2 from human duodenal juice

Phospholipase A2 (EC 3.1.1.4) was purified from delipidated human duodenal juice by hydrophobic and cation exchange chromatography, followed by molecular sieving on an HPLC column. The resulting enzyme preparation of phospholipase A2 had a molecular weight of 14 kDa, a specific activity of 2000 U/mg protein, and an N-terminal amino acid sequence which was characteristic for human pancreatic phospholipase A2.     

Subunit structure of the rat alpha-macroglobulin proteinase inhibitors

Rats produce 2 alpha-macroglobulin (alpha M) proteinase inhibitors, the alpha 1 M, normally found in the plasma, and the alpha 2 M, an acute phase protein. The alpha-macroglobulins were purified from the plasma of rats with adjuvant arthritis by polyethylene glycol precipitation, chromatography on a Zn2+ affinity column, and filtration on Sephacryl S-300 superfine. Comparison of the purified proteins on sodium dodecyl sulfate polyacrylamide gel electrophoresis following reduction reveals a 185 000 Da subunit for rat alpha 2 M identical to the human alpha 2 M, but a 167 000 plus a 38 000 Da subunit for rat alpha 1 M. Heat/alkali treatment (pH 11, 37 degrees C for 45 min) prior to reduction results in the appearance of 125 000 Da and 60 000 Da components from rat alpha 2 M analogous to the pattern of human alpha 2 M. In contrast, alpha 1 M showed in addition to the 125 000 Da band (and the unaltered 38 000 Da band), two bands of approx. 25 000 Da. Incubation with trypsin (approximately 1 mol/mol alpha M) prior to reduction causes formation of approximately 90 000 Da components from both rat inhibitors and the human alpha 2 M. The data suggests that only rat alpha 2 M and not rat alpha 1 M is structurally homologous to human alpha 2 M.     

Purification,characterization and molecular cloning of Vibrio fluvialis hemolysin

Hemolysin of Vibrio fluvialis (VFH) was purified from culture supernatants by ammonium sulfate precipitation and successive column chromatographies on DEAE-cellulose and Mono-Q. N-terminal amino acid sequences of the purified VFH were determined. The purified protein exhibited hemolytic activity on many mammalian erythrocytes with rabbit erythrocytes being the most sensitive to VFH. Activity of the native VFH was inhibited by the addition of Zn2+, Ni2+, Cd2+ and Cu2+ ions at low concentrations. Pores formed on rabbit erythrocytes were approximately 2.8-3.7 nm in diameter, as demonstrated by osmotic protection assay. Nucleotide sequence analysis of the vfh gene revealed an open reading frame (ORF) consisting of 2200 bp which encodes a protein of 740 amino acids with a molecular weight of 82 kDa. Molecular weight of the purified VFH was estimated to be 79 kDa by SDS-PAGE and N-terminal amino acid sequence revealed that the 82 kDa prehemolysin is synthesized in the cytoplasm and is then secreted into the extracellular environment as the 79 kDa mature hemolysin after cleavage of 25 N-terminal amino acids. Deletion of 70 amino acids from the C-terminus exhibited a smaller hemolytic activity, while deletion of 148 C-terminal amino acids prevented hemolytic activity.     

Metal ion affinity adsorption of a Zn(II)-transport protein present in maternal plasma during lactation: Structural characterization and identification as histidine-rich glycoprotein

A high-affinity Zn(II)-binding protein has been purified to homogeneity (880-fold) from the plasma of lactating women by a single affinity adsorption step on columns of tris(carboxymethyl)ethylenediamine (TED)-agarose loaded with Zn(II) ions. Purity was evaluated by high-performance reverse-phase (phenyl) chromatography and by silver staining after SDS-polyacrylamide gradient gel electrophoresis. The mass of denatured Zn(II)-binding protein was estimated by SDS-polyacrylamide gradient gel electrophoresis to be 75 kDa under both reducing and nonreducing conditions; by matrix-assisted uv laser desorption time-of-flight mass spectrometry the purified protein mass was determined to be 66 kDa. The amino acid composition revealed a high content of His (13 mol%) and Pro (12 mol%). N-terminal amino acid sequence analysis (50 residues) identified the purified protein as histidine-rich glycoprotein (HRG). Immunoblots demonstrated the absence of fragments in the purified product. An enzyme-linked immunosorbent assay was developed; a 75% recovery of intact HRG from the immobilized Zn(II) ion affinity column was documented. The circular dichroism spectra for the purified human HRG in the far uv (260-178 nm) were similar to those published for human and rabbit serum HRG. These results demonstrate that TED-immobilized Zn(II) ions can be used as a new and efficient method for the isolation of structurally intact human plasma HRG.     

Purification and characterization of alpha 1-thiol proteinase inhibitor and its identity with kinin- and fragment 1.2-free high molecular weight kininogen

1. alpha 1-Thiol proteinase inhibitor (alpha 1 TPI) purified from outdated human plasma was a glycoprotein with Mr 83,000 and was composed of heavy and light chains held together with a disulfide bond. 2. The data on amino acid composition, amino terminal sequence of the light chain and carboxyl terminal sequences of the heavy and light chains indicate that alpha 1 TPI is identical with kinin- and fragment 1.2-free HMW kininogen. 3. Purified human plasmin generated a derivative having the same molecular weight (Mr 83,000), same subunit structure (heavy and light chains) and same inhibitory capacity as alpha 1 TPI from HMW kininogen and kinin-free HMW kininogen. This indicated the possibility that alpha 1 TPI is derived from HMW kininogen by plasmin.     

Isolation and partial characterization of a novel basement membrane collagen

A guanidine-HCl extraction of lens capsule basement membrane dissolves collagenous material. This material was fractionated on an Agarose A-5M column. Fractions 1, 2 and 3 were further purified and partially characterized immunochemically and by amino acid analysis. Fraction 3 has a molecular weight of 55,000 when compared with collagen type I standard. The CNBr peptide pattern and composition of fraction 3 are different from those of alpha 1 (IV) 95K and alpha 2 (IV) 95K chains. The results described suggest the presence of a new chain in lens capsule basement membrane.     

Identification of gp350 as the viral glycoprotein mediating attachment of Epstein-Barr virus (EBV) to the EBV/C3d receptor of B cells: sequence homology of gp350 and C3 complement fragment C3d.

The major Epstein-Barr virus (EBV) envelope glycoprotein, gp350, was purified from the B95-8 cell line and analyzed for its ability to mediate virus attachment to the isolated EBV/C3d receptor (CR2) of human B lymphocytes. Purified gp350 and EBV, but not cytomegalovirus, exhibited dose-dependent binding to purified CR2 in dot blot immunoassays. Binding was inhibited by certain monoclonal antibodies to CR2 and to gp350. Liposomes bearing incorporated gp350 bound to CR2-positive B-cell lines but not to CR2-negative lines. Liposome binding was also inhibited by the OKB7 anti-CR2 monoclonal antibody. A computer-generated comparison of the deduced gp350 amino acid sequence with that of the human C3d complement fragment revealed two regions of significant primary sequence homology, a finding which suggests that a common region on these two unrelated proteins may be involved in CR2 binding.     

The osteoclast functional antigen, implicated in the regulation of bone resorption, is biochemically related to the vitronectin receptor

We have defined the structure of the Osteoclast Functional Antigen (OFA) by immunological and biochemical means. OFA is an abundant surface antigen in human and animal osteoclasts and has been characterized previously by monoclonal antibodies 13C2 and 23C6, one of which mimicks the inhibitory activity of calcitonin on osteoclastic bone resorption. By the following criteria we show that OFA is a member of the integrin family of extracellular matrix receptors and is identical, or at least highly related, to the vitronectin receptor (VNR) previously isolated from placenta and melanoma cells. Immunoprecipitation analysis demonstrates that OFA from osteoclasts and a monkey kidney cell line Vero is a heterodimeric molecule of 140 kD (alpha chain) and 85 kD (beta chain) under nonreducing conditions; on reduction at least one low molecular mass (alpha') species (of approximately 30-kD size) is released, resulting in a 120/100-kD dimer. Immunoblots of OFA isolated from osteoclasts and Vero cells and VNR purified from placenta and probed with heterosera to OFA and monoclonal antibodies to platelet gp111a (VNR beta chain) show immunological cross- reactivity between the alpha chains of OFA and VNR and the use of gp111a as a beta chain by both. OFA from Vero cells binds to an Arg-Gly- Asp containing peptide (GRGDSPPK) isolating a heterodimer recognized by anti-OFA monoclonal antibodies, 13C2 and 23C6. Immunohistochemical analysis showed a similar tissue distribution in humans for the antigen recognized by anti-OFA antibodies, a monoclonal antibody, LM142, raised to melanoma VNR, polyclonal antibodies to the placental VNR and a monoclonal antibody to the presumptive VNR beta chain, platelet glycoprotein 111a. Finally, NH2 terminal amino acid sequencing showed that the amino-terminus of the monkey alpha chain was identical in the 12 assigned residues to that of human VNR alpha chain. The beta chain sequence of OFA differed at least 1 (and up to 4) positions from platelet gp111a (VNR beta) in the first 18 amino acids sequenced. These, and other, data provide the first indication of a function for the VNR and suggest that cell-cell and cell-extracellular matrix interactions involving integrins may play an important role in bone physiology.     

Studies on camel hemoglobin. 1. Physico-chemical properties and some structural aspects of camel hemoglobin (Camelus dromedarius).

Hemoglobin from an adult camel (Camelus dromedarius) was prepared from the red cell lysate by CM- and DEAE-cellulose chromatography. The purified hemoglobin showed a lesser mobility on starch gel electrophoresis at pH 8.5 than that of human hemoglobin C. Native camel hemoglobin contains 95-99% alkali-resistant hemoglobin and in soluble in 2.94 M K2HPO4/KH2PO4 buffer. Different forms of camel hemoglobin show similar ammonium sulfate precipitation curves. Indirect evidence for the stability of camel hemoglobin solutions was obtained from several sources. Spontaneous met-hemoglobin formation is extremely slow and minimal quantities of degradation products appear on starch gel electrophoresis and on chromatographic separation. The alpha and beta chains of camel hemoglobin A were separated on a CM-23 column by the use of a pyridine formate gradient. Large peptide fragments were obtained by tryptic digestion of maleylated alpha and beta chains. The N-terminal structure of the alpha and beta chains and of tryptic maleylated peptides derived from alpha and beta chains are presented. Between adult camel hemoglobin and adult human hemoglobin six amino acid differences in the N-terminal 20 amino acid residues of the alpha chain, at residues: 4, 5, 12, 14, 17, and 19; eight amino acid substitutions were found in the beta chain at positions: 4, 5, 6, 9, 12, 13, 16, and 19. Substitutions at alpha5 Ala leads to Lys, and beta19 Asn leads to Lys, increase the net positive charge of camel hemoglobin by two, while other substitutions result in no charge differences. The molecular basis of the stability of camel adult hemoglobin is discussed.     

Copurification of small heat shock protein with alpha B crystallin from human skeletal muscle.

Immunoreactive alpha B crystallin and a 28-kDa protein in an extract of human pectoral muscle were precipitated by (NH4)2SO4 at 40% saturation, and coeluted during column chromatography on DEAE-Sepharose and on Bio-Gel A-5m. The two proteins were separated on a column of S-Sepharose HP in the presence of 7 M urea. Further chromatography of each of the two resultant fractions on a column of Superdex 75 pg and on a TSK-SP 5PW column in the presence of urea yielded preparations of alpha B crystallin and the 28-kDa protein each of which gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final preparation of 28-kDa protein contained at least two subtypes, which were separable on the TSK-SP column. However, fragmentation patterns of the two major 28-kDa proteins after digestion with endoproteinase Asp-N were identical. Amino acid sequences of peptides formed by cleavage of the purified 28-kDa protein and alpha B crystallin were identical to those of particular regions of the deduced amino acid sequences of human small heat shock protein (HSP28) and lens alpha B crystallin, respectively. Using an immunoassay method, with antibodies raised in rabbits, we found that HSP28 was present in all human tissues tested and at high levels (greater than 1 micrograms/mg protein) in the heart and other tissues composed of striated and smooth muscles. HSP28, found with alpha B crystallin, in extracts of several human and bovine tissues was trapped on and coeluted with alpha B crystallin from an affinity column prepared with antibodies against alpha B crystallin. This result suggests that the two proteins are associated in cells.