Human methylamine-treated complement C3 (C3-MA) and C3b (C3b-MA) have been crystallized using ammonium sulfate as precipitant. The crystals of the two compounds are morphologically indistinguishable though they belong to different space groups. We show that only minor alterations in packing are responsible for the change in space group. Crystals of C3-MA are tetragonal [P4(1(3))22, a = b = 135, c = 610 A] with two molecules per asymmetric unit. Crystals of C3b-MA are also tetragonal [P4(1(3))2(1)2, a = b = 191, c = 610 A] with four molecules per asymmetric unit. The maximum diffraction observed is 7.7 A at cryogenic temperature using synchrotron radiation.

Journal Article↗

PubMed1994

Structural integrity of the membrane domains in extensively trypsinized Na,K-ATPase from shark rectal glands.

Removal of extramembranous portions of the integral membrane protein Na,K-ATPase from shark salt glands by trypsin in the presence of Rb+ (a K+ congener) preserves the intramembranous association of the remaining membrane-spanning tryptic peptides. This is evidenced from comparison of the rotational mobility of native and trypsinized Na,K-ATPase using saturation transfer electron spin resonance spectroscopy (ESR) and from study of the lipid-protein interactions using conventional ESR spectroscopy. The interface between the lipids and the intramembranous domains is conserved on removal of the extramembranous parts of the protein, since the population of motionally restricted boundary lipids remains essentially the same in the native and trypsinized preparations. The ability to occlude Rb+ is also retained by the trypsinized membranes, as previously observed with pig kidney Na,K-ATPase. A 19-kDa fragment remaining when Na,K-ATPase is trypsinized in the presence of Rb+ is degraded further when the trypsinization is carried out in the presence of Na+ instead of Rb+. The rotational mobility of the tryptic fragments in the Na(+)-trypsinized membranes is lower than for the Rb(+)-trypsinized membranes, indicating rearrangement of the peptides. In addition, occlusion capacity is lost when trypsinization is carried out in Na+, suggesting a correlation between structure and function in the trypsinized membranes. The sequences of four membrane-spanning tryptic fragments of shark Na,K-ATPase are found to be almost identical to corresponding sequences in pig kidney Na,K-ATPase.

Amino Acid Sequence↗

PubMed1994

Receptor-binding domain of human alpha 2-macroglobulin. Expression, folding and biochemical characterization of a high-affinity recombinant derivative.

A recombinant version of the receptor binding domain (RBDv) of human alpha 2-macroglobulin (alpha 2M) has been expressed in E. coli and refolded using a novel iterative procedure. RBDv (Val1299-Ala1451) is extended by 15 residues at the N-terminal side of the Lys1313-Glu papain cleavage site in human alpha 2M. RBDv contains the intra-chain bridge Cys1329-Cys1444 and is soluble and monomeric. Competition experiments with 125I-labelled methylamine-treated alpha 2M reveal that RBDv binds to the placental receptor for transformed alpha 2M with a Kd of 8 nM, i.e. the binding affinity of RBDv is of the same order of magnitude as the intrinsic affinity for binding of one domain in transformed alpha 2M to one receptor molecule.

Amino Acid Sequence↗

PubMed1994

Primary structure of bovine alpha 2-antiplasmin.

The primary structure of bovine alpha 2-antiplasmin (alpha 2AP) has been determined from cDNA and partial peptide sequencing. Mature bovine alpha 2AP contains 470 residues and is 6 residues longer than human alpha 2AP. Alignment of the two protein sequences show that 81% of their amino acid residues are identically located. Bovine alpha 2AP has 5 N-linked carbohydrate groups, of which four are found in human alpha 2AP (Asn105, 274, 288 and 295). Asn227 is the fifth carbohydrate attachement site in bovine alpha 2AP. The 3 Cys residues of bovine alpha 2AP are present as an unpaired residue (Cys131) and as a pair in a disulfide bridge (Cys49-Cys122). The assignment of the bridge in bovine alpha 2AP is at variance with the previous assignment of the two disulfide bridges in human alpha 2AP [Lijnen, H.R. et al. (1987) Eur. J. Biochem. 166, 565-574].

Amino Acid Sequence↗

PubMed1994

Localization of disulfide bridges and free sulfhydryl groups in human eosinophil granule major basic protein.

Eosinophil granule major basic protein (117 residues) is known to contain free sulfhydryl groups. Here we have located in the amino acid sequence the half-cystine residues present as cysteine, and identified those engaged in disulfide bridges. Of the 9 half-cystine residues, 5 are unpaired cysteines (Cys2, Cys23, Cys42, Cys64, and Cys96), while 4 form disulfides (Cys20-Cys115, and Cys92-Cys107).

Amino Acid Sequence↗

PubMed1994

Amino acid sequence of human pregnancy-associated plasma protein-A derived from cloned cDNA.

The amino acid sequence of human pregnancy-associated plasma protein-A (PAPP-A), a component of the circulating complex with the proform of eosinophil major basic protein (proMBP), has been determined from partial protein sequencing and from sequencing of cloned cDNA. The PAPP-A monomer contains 1547 amino acid residues, but is derived from a larger precursor of placental origin. PAPP-A contains 82 Cys residues, which are all bridged, 14 putative sites for N-glycosylation, and 7 putative sites for attachment of glycosaminoglycan groups. The C-terminal part of PAPP-A contains 5 approximately 60-residue motifs related to the short consensus repeats of complement proteins and selectins. The SCRs presently known can be grouped into three classes: complement-type, class I; selectin-type, class II; PAPP-A-type, class III. PAPP-A further contains three approximately 26-residue motifs, related to the lin-notch motifs of proteins regulating early tissue differentiation, and, in addition, a putative Zn2+ binding site similar to that found in many metalloproteinases has been identified. Apart from these features, the PAPP-A sequence is not related to other known protein sequences.

Amino Acid Sequence↗

PubMed1994

Receptor-mediated endocytosis of plasminogen activators and activator/inhibitor complexes.

Recent findings have elucidated the mechanism for clearance from the extracellular space of the two types of plasminogen activators, urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA), and their type-1 inhibitor (PAI-1). Activator/PAI-1 complexes and uncomplexed t-PA bind to the multi-ligand receptors alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR) and epithelial glycoprotein 330 (gp330). These receptors mediate endocytosis and degradation of u-PA/PAI-1 complex bound to the glycosyl phosphatidyl inositol-anchored urokinase receptor (u-PAR) on cell surfaces, and participate, in cooperation with other receptors, in hepatic clearance of activator/PAI-1 complexes and uncomplexed t-PA from blood plasma. The alpha 2MR- and gp330-mediated endocytosis of a ligand (u-PA/PAI-1 complex) initially bound to another receptor (u-PAR) is a novel kind of interaction between membrane receptors. Binding to alpha 2MR and gp330 is a novel kind of molecular recognition of serine proteinases and serpins.

Amino Acid Sequence↗

PubMed1994

Biochemical properties, activities, and presence in biologic fluids of eosinophil granule major basic protein.

The existence of a proform of MBP is predicted from the sequence of MBP cDNA clones. ProMBP has been purified from the supernatants of CHO cells transfected with cDNA encoding prepro MBP. Purification involved heparin-Sepharose affinity purification followed by two sequential size fractionation steps over Sephadex G-100 and yielded proMBP with a molecular mass of 33 kd. Recombinant proMBP from the heparin-Sepharose column was subjected to isoelectric focusing followed by SDS-PAGE and Western blot analysis. The results indicated that most of the 33 kd form of proMBP focused predominantly between pI 4.2 and 5.1, with a major peak at a pI of approximately 4.9. Analyses of the carbohydrates associated with the purified 33 kd form of recombinant proMBP indicated the addition of 4856 to 5150 Da by carbohydrates characteristic of the complex type. Consistent with the hypothesis that the function of the propiece is to neutralize MBP toxicity during granule processing, proMBP lacked MBP cytostimulatory properties and actually blocked the effect of MBP in two different systems, basophil histamine release and neutrophil activation. In addition, as a measure of toxicity, proMBP did not inhibit protein synthesis, whereas MBP markedly reduced protein synthesis. The mechanisms by which MBP exerts its actions both as a cytostimulant and as a toxin are not known; however, it is known that cationic MBP readily reacts with acidic lipids. Using artificial liposomes as targets, MBP caused a disordering of the lipid bilayer membrane, resulting in fusion and lysis. Therefore, MBP may act both as a cytostimulant and as a toxin because of its marked cationicity and its ability to disorder lipid membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Amino acid sequence of hen ovomacroglobulin (ovostatin) deduced from cloned cDNA.

The complete amino acid sequence of the hen ovomacroglobulin (ovostatin) subunit has been determined from cDNA and partial peptide sequence analysis. Ovostatin is a tetrameric member of the alpha-macroglobulin (alpha M) family of proteins. The 4715 nt ovostatin cDNA encodes a 36- or a 16-residue signal peptide and a 1437-residue mature protein (162.2 kDa). At the protein level the overall score of sequence identity between ovostatin and mammalian alpha Ms is 39-44%, indicating an early divergence from the line leading to the mammalian alpha Ms. Ovostatin contains 56 mol glucosamine per mol subunit, and 12 of its Asn-residues are likely to be N-glycosylated. Including carbohydrate, the size of the ovostatin subunit is approx. 185 kDa. The ovostatin subunit is predicted to contain 12 intrachain disulfide bridges, and two subunits are predicted to be disulfide bound by two interchain bridges. One Cys residue may be unpaired or participate in dimer formation as a third interchain disulfide bridge. Ovostatin contains a unique 40-residue bait region. In contrast to other alpha Ms, ovostatin contains no internal beta-Cys-gamma-Glu thiol ester, as a result of a Cys-to-Asn replacement (TGC or TGT to AAT), but the Gln-moiety of the thiol ester is preserved. By comparing the sequences of the receptor binding domain in alpha Ms with the corresponding region of ovostatin possible determinants for receptor recognition of mammalian alpha Ms are proposed.

Amino Acid Sequence↗

PubMed1994

Location and nature of carbohydrate groups in proform of human major basic protein isolated from pregnancy serum.

From human pregnancy serum we have isolated the proform of eosinophil major basic protein (proMBP), which forms a complex with pregnancy-associated plasma protein-A (PAPP-A), PAPP-A/proMBP. It is shown that proMBP contains O-linked glycan bound to Ser-24, Thr-25 (fully substituted), and to Thr-23 and Thr-34 (partially substituted). N-linked glycan is bound to Asn-86 and O-linked glycosaminoglycan is bound to Ser-62 (both fully substituted). From the RP-HPLC elution profile and mass spectra of tryptic peptides it is found that proMBP is extremely heterogeneous with respect to glycosylation.

Amino Acid Sequence↗

PubMed1994

Localization of pregnancy-associated plasma protein-A and colocalization of pregnancy-associated plasma protein-A messenger ribonucleic acid and eosinophil granule major basic protein messenger ribonucleic acid in placenta.

BACKGROUND: The human eosinophil granule major basic protein (MBP), a 13.8 kilodalton cationic polypeptide constituting the core of the eosinophil granule, is cytotoxic to parasites and numerous mammalian cells. Concentrations of a molecule immunochemically similar to eosinophil granule MBP are present in maternal plasma, and MBP mRNA has been localized to placental X cells by in situ hybridization. Eosinophil granule MBP is initially translated as a nontoxic precursor (proMBP), containing a 9.9 kilodalton acidic pro-portion that is believed to neutralize MBP toxicity. Recent analyses of sera from pregnant women have revealed that pregnancy-associated plasma protein-A (PAPP-A), previously thought to be a homotetramer of PAPP-A subunits, is actually composed of PAPP-A subunits bound by disulfide bonds to equimolar amounts of proMBP molecules to form a complex, PAPP-A/proMBP. In addition, the PAPP-A subunit nucleotide and deduced amino acid sequence have been determined from cloned cDNA. The PAPP-A monomer found in plasma contains 1547 amino acid residues. EXPERIMENTAL DESIGN: Because of the new evidence that PAPP-A is complexed with proMBP, previous studies on the localization of PAPP-A using antibodies to PAPP-A must be questioned. To determine the localization of the PAPP-A subunit, immunofluorescence was performed on normal placental tissues using proMBP absorbed anti-PAPP-A antibody. Furthermore, the expression of PAPP-A mRNA was investigated by in situ hybridization. RESULTS: Immunofluorescence staining with proMBP absorbed anti-PAPP-A antibody showed that PAPP-A is localized to placental septa, anchoring villi, and the syncytia of chorionic villi, whereas MBP is localized only to septa and anchoring villi. By in situ hybridization, PAPP-A mRNA is detected in placental X cells and syncytiotrophoblasts, but MBP mRNA is localized only to placental X cells. CONCLUSIONS: The presence of PAPP-A mRNA and PAPP-A subunit protein in placental X cells and syncytiotrophoblasts indicates that both X cells and syncytiotrophoblasts synthesize the PAPP-A subunit, whereas only X cells synthesize proMBP.

Blood Proteins↗

PubMed1993

Alpha 2-macroglobulin-proteinase complexes, plasminogen activator inhibitor type-1-plasminogen activator complexes, and receptor-associated protein bind to a region of the alpha 2-macroglobulin receptor containing a cluster of eight complement-type repeats.

A region containing sites for ligand binding was localized in the 4525-amino acid residue alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) by ligand- and immunoblotting of proteinase and CNBr digests of the purified human placental protein. 125I-Labeled rat alpha 1-macroglobulin light chain, urokinase-plasminogen activator inhibitor type-1 complex, and alpha 2MR-associated protein all bound to a 75-kDa CNBr-generated fragment (68 kDa after deglycosylation). In addition to the three ligands, the fragment bound a novel monoclonal antibody reacting in the region defined by amino acid residues 1165-1246 as determined by binding to recombinant fragments of alpha 2MR/LRP. The positions of methionine residues in alpha 2MR/LRP suggested that the ligand-binding CNBr fragment contained three disulfide-linked peptides comprising the residues 776-1399. This origin was confirmed by partial amino acid sequencing of the electroblotted fragment and polypeptides generated by reduction of the fragment. The identified region represents 13.6% of the molecular mass (nonglycosylated) of alpha 2MR/LRP and contains one of three large clusters of complement-type repeats.

Amino Acid Sequence↗

PubMed1993

Circulating human pregnancy-associated plasma protein-A is disulfide-bridged to the proform of eosinophil major basic protein.

A previously unrecognized association between pregnancy-associated plasma protein-A (PAPP-A) and the proform of eosinophil major basic protein (proMBP) is demonstrated. PAPP-A isolated from pooled pregnancy serum is shown to be a disulfide-bridged complex with proMBP (PAPP-A/proMBP) in which the subunits of the constituents are present in a 1:1 molar ratio. The results are the outcome of analysis of tryptic and CNBr/tryptic peptides from PAPP-A/proMBP, sequence analysis of intact and reduced and carboxymethylated PAPP-A/proMBP, and reaction with monoclonal antibodies directed against MBP and its proform. In addition, it is shown that commercial polyclonal anti-PAPP-A is polyspecific, also reacting with MBP.

Amino Acid Sequence↗

PubMed1993

Evidence from sequence analysis that hen egg-white ovomacroglobulin (ovostatin) is devoid of an internal beta-Cys-gamma-Glu thiol ester.

53 residues of the internal sequence from the proteinase-binding hen egg-white ovostatin have been determined. The stretch corresponds to residues 945-997 of human alpha 2-macroglobulin. The degree of conservation of residues of the two stretches is approx. 74%. Cys-949, being one constituent of the internal thiol ester site of members of the family of proteins related to alpha 2-macroglobulin, is an Asn-residue in hen egg-white ovostatin, but the other constituent, Gln-952, is preserved. The Cys-to-Asn substitution forms the chemical basis for the lack of thiol esters in hen egg-white ovostatin.

Amino Acid Sequence↗