Salt-induced refolding in different domains of partially folded bovine serum albumin

In our earlier communication on urea denaturation of bovine serum albumin (BSA), we showed significant unfolding of domain III along with domain I prior to intermediate formation around 4.6-5.2 M urea based on the binding results of domain specific ligands:chloroform, bilirubin and diazepam for domains I, II and III, respectively. Here, we present our results on the salt-induced refolding of the two partially folded states of BSA obtained at 4.5 M urea and at pH 3.5, respectively. Both these states were characterized by significant unfolding of both domains I and III as indicated by decreased binding of chloroform and diazepam, respectively. Salt-induced stabilization of partially folded states of BSA was accompanied by nearly complete refolding of both domains I and III as the binding isotherms of chloroform and diazepam obtained in presence of approximately 1.0 M KCl were nearly identical to that obtained with native BSA at pH 7.4. From these observations, it can be concluded that the anion binding sites on serum albumin are not only confined to domain III (C-terminal region) but few sites are also present on domain I (or N-terminal region) of the protein.     

Detection and localization of a cytoplasmic domain on the beta-subunit of Na+,K+-ATPase. A monoclonal antibody study

A monoclonal antibody directed against the beta-subunit of dog kidney Na+,K+-ATPase was generated. Immunoblots demonstrate that monoclonal antibody III 18A binds exclusively to the denaturated beta-subunit. Binding experiments with membranes and whole cells reveal that III 18A binds to membranes but not to whole cells, indicating that the antibody binds to a cytoplasmic domain on the native beta-subunit. To localize the antibody-binding epitope, purified membrane-bound enzyme was fragmented by protease treatment. Tryptic digestion yields a 30-kDa fragment of the beta-subunit, which still retains the binding capacity for the antibody. Thus III 18A probably does not bind to the NH2-terminal segment of the protein. On the other hand, fragmentation of the beta-subunit with low concentrations of papain, which is known to yield a 40-kDa NH2-terminal and a 16-kDa COOH-terminal fragment, results in a complete loss of III 18A binding. These results suggest that the antibody-binding epitope is localized at or near a papain cleavage site on the COOH-terminal part of the beta-subunit. This is inconsistent with a structure model of the beta-subunit containing only a single transmembrane hydrophobic segment with a cytoplasmic NH2-terminal portion, but agrees quite well with a hypothetical structure with four intramembrane segments.     

A structural model of the acetylcholine receptor channel based on partition energy and helix packing calculations.

A structural model of the transmembrane portion of the acetylcholine receptor was developed from sequences of all its subunits by using transfer energy calculations to locate transmembrane alpha-helices and to calculate which helical side chains should be in contact with water inside the channel, with portions of other transmembrane helices, or with lipid hydrocarbon chains. "Knobs-into-holes" side chain packing calculations were used with other factors to stack the transmembrane alpha-helices together. In the model each subunit has the following structures in order along the sequence from the NH2 terminus: a large extracellular domain of undetermined structure, a short apolar alpha-helix that lies on the extracellular lipid surface of the membrane; three apolar transmembrane alpha-helices (I, II, and III), a cytoplasmic domain of undetermined structure, an amphipathic transmembrane alpha-helix (L) that forms the channel lining, a short extracellular alpha-helix, another apolar transmembrane alpha-helix (IV), and a small cytoplasmic domain formed by the COOH-terminal end of the chain. Three concentric layers form the pore. A bundle of five amphipathic L helices forms the channel lining. This bundle is surrounded by a bundle of 10 alternating II and III helices. Helices I and IV cover portions of the outer surface of the bundle formed by helices II and III. Positions of disulfide bridges are predicted and a mechanism for opening and closing conformational changes is proposed that requires tilting transmembrane helices and possibly a thiol-disulfide interchange reaction.     

The upper cytokine-binding module and the Ig-like domain of the leukaemia inhibitory factor (LIF) receptor are sufficient for a functional LIF receptor complex.

To elucidate the function of the two cytokine-binding modules (CBM) of the leukemia inhibitory factor receptor (LIFR), receptor chimeras of LIFR and the interleukin-6 receptor (IL-6R) were constructed. Either the NH(2)-terminal (chimera RILLIFdeltaI) or the COOH-terminal LIFR CBM (chimera RILLIFdeltaII) were replaced by the structurally related CBM of the IL-6R which does not bind LIF. Chimera RILLIFdeltaI is functionally inactive, whereas RILLIFdeltaII binds LIF and mediates signalling as efficiently as the wild-type LIFR. Deletion mutants of the LIFR revealed that both the NH(2)-terminal CBM and the Ig-like domain of the LIFR are involved in LIF binding, presumably via the LIF site III epitope. The main function of the COOH-terminal CBM of the LIFR is to position the NH(2)-terminal CBM and the Ig-like domain, so that these can bind to LIF. In analogy to a recently published model of the IL-6R complex, a model of the active LIFR complex is suggested which positions the COOH-terminal CBM at LIF site I and the NH(2)-terminal CBM and the Ig-like domain at site III. An additional contact is postulated between the Ig-like domain of gp130 and the NH(2)-terminal CBM of the LIFR.     

Unfolding pathways of human serum albumin: evidence for sequential unfolding and folding of its three domains

Human serum albumin (HSA) contains three alpha-helical domains (I-III). The unfolding process of these domains was monitored using covalently bound fluorescence probes; domain I was monitored by N-(1-pyrene)maleimide (PM) conjugated with cys-34, domain II was monitored by the lone tryptophan residue and domain III was followed by p-nitrophenyl anthranilate (NPA) conjugated with Tyrosine-411 (Tyr-411). Using domain-specific probes, we found that guanidium hydrochloride-induced unfolding of HSA occurred sequentially. The unfolding of domain II preceded that of domain I and the unfolding of domain III followed that of domain I. In addition, the domains I and III refolded within the dead time of the fluorescence recovery experiment while the refolding of domain II occurred slowly. The results suggest that individual domain of a multi-domain protein can fold and unfold sequentially.     

Structural and functional studies of the dnaB protein using limited proteolysis. Characterization of domains for DNA-dependent ATP hydrolysis and for protein association in the primosome

Two separable structural domains were identified in the Escherichia coli dnaB protein (Mr = 52,000) by partial proteolytic cleavage under nondenaturing conditions. The hydrolysis of dnaB protein by trypsin proceeded in two distinct stages in the presence of ATP or ADP. In the first stage, 14 amino acid residues at the NH2-terminal end were removed and dnaB protein was converted into a fragment with a molecular weight of 50,000 (Fragment I). Fragment I retained about 60% of the original activity in priming DNA replication and was fully active in DNA-dependent ATPase activity. In the second stage, Fragment I was further cleaved into two separable polypeptides with molecular weights of 33,000 (Fragment II) and 12,000 (Fragment III), respectively. Fragment II, as a hexamer, retained DNA-dependent ATPase activity comparable to the intact protein but was totally inactive in priming DNA replication. No known activity of dnaB protein was detected in Fragment III alone. NH2 termini of Fragments I and III and COOH termini of dnaB protein and Fragment II were identical indicating that Fragments III and II were located at the NH2 and COOH termini of Fragment I, respectively. These results indicate that dnaB protein is composed of at least two distinct domains. 1) Fragment III, the rigid domain, is essential for protein interaction, i.e. association with dnaC protein and primase in priming DNA replication in the primosome. 2) A 14-amino acid residue fragment, at the NH2-terminal end adjacent to Fragment III, probably required to stabilize the protein interaction involved in priming DNA replication. 3) Fragment II, the flexible COOH-terminal domain, contains the active sites for DNA binding, ATP binding, and protein oligomerization. Fragment II is cleaved by trypsin at many sites in the absence of ATP or ADP ligands. The rate of conversion of Fragment I into the yield of Fragments II and III was decreased approximately by 2 orders of magnitude by changing the ligand from ADP to the nonhydrolyzed ATP analog, adenosine 5'-O-(3-thiotriphosphate). These results indicate that the conformation of the COOH-terminal domain in the dnaB protein is stabilized by ATP or ADP. Such a nucleotide-induced conformational change was also demonstrated by circular dichroism spectroscopy. Moreover, the data suggest that the conformation of the dnaB protein complexed with adenosine 5'-O-(3-thiotriphosphate) is different from that complexed with ADP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential effects of parathyroid hormone fragments on collagen gene expression in chondrocytes

The effect of parathyroid hormone (PTH) in vivo after secretion by the parathyroid gland is mediated by bioactive fragments of the molecule. To elucidate their possible role in the regulation of cartilage matrix metabolism, the influence of the amino-terminal (NH2-terminal), the central, and the carboxyl-terminal (COOH-terminal) portion of the PTH on collagen gene expression was studied in a serum free cell culture system of fetal bovine and human chondrocytes. Expression of alpha1 (I), alpha1 (II), alpha1 (III), and alpha1 (X) mRNA was investigated by in situ hybridization and quantified by Northern blot analysis. NH2- terminal and mid-regional fragments containing a core sequence between amino acid residues 28-34 of PTH induced a significant rise in alpha1 (II) mRNA in proliferating chondrocytes. In addition, the COOH-terminal portion (aa 52-84) of the PTH molecule was shown to exert a stimulatory effect on alpha1 (II) and alpha1 (X) mRNA expression in chondrocytes from the hypertrophic zone of bovine epiphyseal cartilage. PTH peptides harboring either the functional domain in the central or COOH-terminal region of PTH can induce cAMP independent Ca2+ signaling in different subsets of chondrocytes as assessed by microfluorometry of Fura-2/AM loaded cells. These results support the hypothesis that different hormonal effects of PTH on cartilage matrix metabolism are exerted by distinct effector domains and depend on the differentiation stage of the target cell.     

Direct evidence for the involvement of domain III in the N-F transition of bovine serum albumin.

The domain III of bovine serum albumin containing residues 377-582 of the protein sequence was isolated and its behaviour in acid solution was studied. The fragment was found to undergo structural transformations over the pH range 3.5-4.5 known to cause N-F transition in serum albumin. On the other hand, an albumin fragment that was devoid of domain III was unable to exhibit such a transition. These results were consistent with a mechanism where N-F transition involves the separation of domain III from the rest of the albumin starts at about pH 4.3 and is completed at pH 3.5.     

Limited pepsin digestion of human plasma albumin.

Limited pepsin digestion of human plasma albumin at pH 3.5 and 0 degrees in the presence of octanoate caused cleavage at residue 307 of the albumin molecule to yield two fragments. Thw two fragments corresponding to the NH2- and the COOH-terminal halves of the molecule were isolated in yields of about 15%. The COOH-terminal fragment is a mixture in which about 85% of the molecules had an additional cleavage at residue 422 of the albumin molecule. The COOH-terminal fragment with the additional cleavage at residue 422 contains two peptides which are linked by a disulfide bridge at residues 391 and 437 of the albumin molecule. Both the NH2- and the COOH-terminal fragment of human albumin showed no detectable binding of octanoate anions, that is, less than 1/170 of the binding constant of the primary site of human albumin. These findings differ from earlier observations on limited pepsin digestion of bovine plasma albumin where the corresponding COOH-terminal fragment had the octanoate-binding activity, about 1/8 of the primary binding constant of bovine albumin, while the NH2-terminal fragment did not. The COOH-terminal fragment of bovine albumin did not have cleavage at residue 422 as in the corresponding fragment of human albumin. However, it is not clear that the loss of octanoate-binding activity of fragment C of human albumin is a direct consequence of the cleavage at residue 422.     

Mannose-binding proteins isolated from rat liver contain carbohydrate-recognition domains linked to collagenous tails. Complete primary structures and homology with pulmonary surfactant apoprotein

Preparations of mannose-binding protein isolated from rat liver contain two distinct but homologous polypeptides. The complete primary structures of both of these polypeptides have been determined by sequencing of peptides derived from the proteins, isolation and sequencing of cDNAs for both proteins, and partial characterization of the gene for one of the proteins. Each polypeptide consists of three regions: (a) an NH2-terminal segment of 18-19 amino acids which is rich in cysteine and appears to be involved in the formation of interchain disulfide bonds which stabilize dimeric and trimeric forms of the protein, (b) a collagen-like domain consisting of 18-20 repeats of the sequence Gly-X-Y and containing 4-hydroxyproline residues in several of the Y positions, and (c) a COOH-terminal carbohydrate-binding domain of 148-150 amino acids. The sequences of the COOH-terminal domains are highly homologous to the sequence of the COOH-terminal carbohydrate-recognition portion of the chicken liver receptor for N-acetylglucosamine-terminated glycoproteins and the rat liver asialoglycoprotein receptor. Each protein is preceded by a cleaved, NH2-terminal signal sequence, consistent with the finding that this protein is found in serum as well as in the liver. The entire structure of the mannose-binding proteins is homologous to dog pulmonary surfactant apoprotein.     

A yeast Golgi E-type ATPase with an unusual membrane topology.

E-type ATPases are involved in many biological processes such as modulation of neural cell activity, prevention of intravascular thrombosis, and protein glycosylation. In this study, we show that a gene of Saccharomyces cerevisiae, identified by similarity to that of animal ectoapyrase CD39, codes for a new member of the E-type ATPase family (Apy1p). Overexpression of Apy1p in yeast cells causes an increase in intracellular membrane-bound nucleoside di- and triphosphate hydrolase activity. The activity is highest with ADP as substrate and is stimulated similarly by Ca (2+), Mg(2+), and Mn(2+). The results also indicate that Apy1p is an integral membrane protein located predominantly in the Golgi compartment. Sequence analysis reveals that Apy1p contains one large NH(2)-terminal hydrophilic apyrase domain, one COOH-terminal hydrophilic domain, and two hydrophobic stretches in the central region of the polypeptide. Although no signal sequence is found at the NH(2)-terminal portion of the protein and no NH(2)-terminal cleavage of the protein is observed, demonstrated by the detection of NH(2)-terminal tagged Apy1p, the NH(2)-terminal domain of Apylp is on the luminal side of the Golgi apparatus, and the COOH-terminal hydrophilic domain binds to the cytoplasmic face of the Golgi membrane. The second hydrophobic stretch of Apy1p is the transmembrane domain. These results indicate that Apylp is a type III transmembrane protein; however, the size of the Apy1p extracytoplasmic NH(2) terminus is much larger than those of other type III transmembrane proteins, suggesting that a novel translocation mechanism is utilized.     

The architecture of the animal fatty acid synthetase complex. IV. Mapping of active centers and model for the mechanism of action

The fatty acid synthetase of animal tissue consists of two subunits, each containing seven catalytic centers and an acyl carrier site. Proteolytic cleavage patterns indicate that the subunit is arranged into three major domains, I, II, and III. Domain I contains the NH2-terminal end of the polypeptide and the catalytic sites of beta-ketoacyl synthetase (condensing enzyme) and the acetyl-and malonyl-transacylases. This domain, therefore, functions as a site for acetyl and malonyl substrate entry into the process of fatty acid synthesis and acts in part as the site of carbon-carbon condensation, resulting in chain elongation. Domain II is the medial domain and contains the beta-ketoacyl and enoyl reductases, probably the dehydratase, and the 4'-phosphopantetheine prosthetic group of the acyl carrier protein site. Domain II, therefore, is designated as the reduction domain where the keto carbon is reduced to methylene carbon by sequential processes of reduction, dehydration, and reduction again. Throughout these processes, the acyl group is attached to the pantetheine-SH of the acyl carrier protein. The latter site is distal to the cysteine-SH of the beta-ketoacyl synthetase, constitutes the 15000-dalton polypeptide at the COOH-terminal end of Domain II, and connects to Domain III. When the growing chain reaches C16 carbon length, the fatty acyl group is released by the thioesterase activity, which is contained in Domain III. A functional model is proposed based on the aforementioned results and the recent evidence that the synthetase subunits are arranged in a head-to-tail fashion, such that the pantetheine-SH of the acyl carrier protein of one subunit and the cysteine-SH of the beta-ketoacyl synthetase of the second subunit are juxtaposed. In this model, a palmitate synthesizing site contains Domain I of one subunit and Domains II and III of the second subunit. Therefore, even though each subunit contains all of the partial activities of the reaction sequence, the actual palmitate synthesizing unit consists of one-half of a subunit interacting with the complementary half of the other subunit.     

Mammalian heterogeneous nuclear ribonucleoprotein complex protein A1. Large-scale overproduction in Escherichia coli and cooperative binding to single-stranded nucleic acids

Characterization of mammalian heterogeneous nuclear ribonucleoprotein complex protein A1 is reported after large-scale overproduction of the protein in Escherichia coli and purification to homogeneity. A1 is a single-stranded nucleic acid binding protein of 320 amino acids and 34,214 Da. The protein has two domains. The NH2-terminal domain is globular, whereas the COOH-terminal domain of about 120 amino acids has low probability of alpha-helix structure and is glycinerich. Nucleic acid binding properties of recombinant A1 were compared with those of recombinant and natural proteins corresponding to the NH2-terminal domain. A1 bound to single-stranded DNA-cellulose with higher affinity than the NH2-terminal domain peptides. Protein-induced fluorescence enhancement was used to measure equilibrium binding properties of the proteins. A1 binding to poly (ethenoadenylate) was cooperative with the intrinsic association constant of 1.5 X 10(5) M-1 at 0.4 M NaCl and a cooperativity parameter of 30. The NH2-terminal domain peptides bound noncooperatively and with a much lower association constant. With these peptides and with intact A1, binding was fully reversed by increasing [NaCl]; yet. A1 binding was much less salt-sensitive than binding by the NH2-terminal domain peptides. A synthetic polypeptide analog of the COOH-terminal domain was prepared and was found to bind tightly to poly-(ethenoadenylate). The results are consistent with the idea that the COOH-terminal domain contributes to A1 binding through both cooperative protein-protein interaction and direct interaction with the nucleic acid.     

Endogenous lectin from cultured soybean cells: exposure of the SB-1 lectin on the cell wall

Digestion of seed soybean agglutinin with V-8 protease yielded seven distinct fragments (Mr 10,000-20,000) that were well-resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Each individual peptide (F1 through F7) was isolated; determination of the amino acid sequence at the NH2-terminal portion of each peptide established its position in the intact polypeptide of soybean agglutinin. The isolated peptides were used as affinity adsorbents to obtain antibodies that bound individual fragments (anti-F1 through anti-F7). These antibody preparations were, in turn, used in immunofluorescence staining of intact cultured soybean (SB-1) cells. Only those antibody preparations that bind to the NH2-terminal portion (residues 1-124) of the intact soybean agglutinin showed significant cell surface labeling. In contrast, the antibody preparations that bound to residues 125-253 failed to bind to intact SB-1 cells. These results suggest that the SB-1 lectin has the NH2-terminal portion of the polypeptide chain exposed and accessible at the cell surface, while the COOH-terminal portion of the same molecule may be masked, either through protein folding or through embedding in the cell wall. Limited digestion of the cell wall polysaccharides by cellulase or pectinase released the majority of the cell surface lectin.     

Ubiquitination of p53 at multiple sites in the DNA-binding domain

The tumor suppressor p53 is negatively regulated by the ubiquitin ligase MDM2. The MDM2 recognition site is at the NH2-terminal region of p53, but the positions of the actual ubiquitination acceptor sites are less well defined. Lysine residues at the COOH-terminal region of p53 are implicated as sites for ubiquitination and other post-translational modifications. Unexpectedly, we found that substitution of the COOH-terminal lysine residues did not diminish MDM2-mediated ubiquitination. Ubiquitination was not abolished even after the entire COOH-terminal regulatory region was removed. Using a method involving in vitro proteolytic cleavage at specific sites after ubiquitination, we found that p53 was ubiquitinated at the NH2-terminal portion of the protein. The lysine residue within the transactivation domain is probably not essential for ubiquitination, as substitution with an arginine did not affect MDM2 binding or ubiquitination. In contrast, several conserved lysine residues in the DNA-binding domain are critical for p53 ubiquitination. Removal of the DNA-binding domain reduced ubiquitination and increased the stability of p53. These data provide evidence that in addition to the COOH-terminal residues, p53 may also be ubiquitinated at sites in the DNA-binding domain.     

Binding of pyridoxal 5'-phosphate to bovine serum albumin and albumin fragments obtained after proteolytic hydrolysis. Localization and nature of the primary PLP binding site.

The binding of pyridoxal 5'-phosphate (PLP) to bovine serum albumin (BSA), and to large BSA fragments obtained after proteolytic hydrolysis, was investigated in order to study the structure of these fragments in relation to the albumin structure itself, and to get information about the PLP binding sites on albumin. From absorbance and circular dichroism spectra, combined with peptide mapping of the tryptic digests of the reduced PLP-protein complexes, it could be concluded that the primary binding site is localized with the NH2-terminal part of the albumin molecule. The COOH-terminal part contains one or more secondary sites. It appeared that in albumin and in the largest NH2-terminal fragment, the environment of the primary binding site is rather apolar in character. However, in the smallest NH2-terminal fragment this site is more exposed to the solvent. This suggests that the part of the peptide chain which is not common in both fragments has a stabilizing effect on the structure around the primary binding site.     

Cleavage of Type II and III collagens with mammalian collagenase: site of cleavage and primary structure at the NH2-terminal portion of the smaller fragment released from both collagens.

Collagenase cleavage of human Type II and III collagens has been studied using a highly purified preparation of rabbit tumor collagenase. Progress of the reactions in solution was followed by viscometry and the results indicated that under the conditions employed Type III collagen molecules were cleaved at approximately five times the rate of Type II molecules. Cleavage products of the reactions were isolated in denatured form by agarose molecular sieve chromatography. The molecular weights and amino acid compositions of the products demonstrated that Type II and III molecules had been cleaved at the characteristic three-quarter, one-quarter locus, giving rise to a large fragment derived from the NH2-terminal portion of the molecule and a smaller fragment representing the COOH-terminal region. The amino acid sequence at the NH2-terminal portion of the smaller fragment derived from Type II collagen was determined to be Ile-Ala-Gly-Gln-Arg, and the corresponding region from Type III collagen was found to have the sequence Leu-Ala Gly-Leu-Arg. These sequences for alpha1(II) and alpha1(III) chains adjacent to the site of collagenase cleavage along with previous data for alpha1(I) and alpha2 chains indicate that the minimum specific sequence required for collagenase cleavage is Gly-Ile-Ala or Gly-Leu-Ala. Inspection of the available sequence data for collagen alpha chains indicates that the latter sequences are found in at least three additional locations at which collagenase cleavage does not occur. Each of the sequences which are apparently not substrates for collagenase, however, are followed by a Gly-X-Hyp sequence. We suggest, then, that a minimum of five residues in collagen alpha chains COOH-terminal to the cleavage site comprise the substrate recognition site.     

Thrombospondins: structure and regulation of expression.

Thrombospondin (TSP) is a large, trimeric, modular glycoprotein that is a major constituent of platelet alpha granules. TSP is also secreted by a wide variety of epithelial and mesenchymal cells in patterns that reflect developmental changes in the embryo and response to injury in the adult. In addition to its role in blood coagulation, TSP has been reported to serve both adhesive and anti-adhesive functions, to foster neurite outgrowth, stimulate and inhibit cell growth and migration, and inhibit angiogenesis. Although this diversity in apparent function can be attributed, in part, to the ability of a single TSP to interact with several different cell-surface receptors, it is now known that the TSPs are encoded by at least three homologous genes in both human and mouse. TSP1, the commonly recognized protein isolated from platelets, is similar to TSP2 in structure. Both proteins contain NH2-terminal, COOH-terminal, and procollagen homology domains, and type I (TSP or properdin), type II (EGF-like), and type III (Ca(2+)-binding) repeats. However, the two TSPs differ in amino acid sequence and in the regulation of their expression. TSP1 is rapidly induced by serum and growth factors. An SRE and a binding site for NF-Y have been shown to mediate the serum response of the human TSP1 gene. On the other hand, TSP2 is far less responsive to serum than TSP1 and lacks the promoter elements that mediate the serum responsiveness of TSP1. TSP3 resembles TSP1 and TSP2 in its COOH-terminal domain and type III repeats, but contains four rather than three type II repeats and lacks type I repeats and a procollagen homology. The NH2-terminal domain of TSP3 also differs from that of either TSP1 or TSP2. All three TSPs demonstrate characteristic patterns of expression in the developing and adult mouse. It is therefore likely that each protein subserves a discrete function. In the future it will be necessary to distinguish among the three TSPs in addressing the function of these proteins.     

Chimeras of hepatic lipase and lipoprotein lipase. Domain localization of enzyme-specific properties.

Chimeric molecules between human lipoprotein lipase (LPL) and rat hepatic lipase (HL) were used to identify structural elements responsible for functional differences. Based on the close sequence homology with pancreatic lipase, both LPL and HL are believed to have a two-domain structure composed of an amino-terminal (NH2-terminal) domain containing the catalytic Ser-His-Asp triad and a smaller carboxyl-terminal (COOH-terminal) domain. Experiments with chimeric lipases containing the HL NH2-terminal domain and the LPL COOH-terminal domain (HL/LPL) or the reverse chimera (LPL/HL) showed that the NH2-terminal domain is responsible for the catalytic efficiency (Vmax/Km) of these enzymes. Furthermore, it was demonstrated that the stimulation of LPL activity by apolipoprotein C-II and the inhibition of activity by 1 M NaCl originate in structural features within the NH2-terminal domain. HL and LPL bind to vascular endothelium, presumably by interaction with cell surface heparan sulfate proteoglycans. However, the two enzymes differ significantly in their heparin affinity. Experiments with the chimeric lipases indicated that heparin binding avidity was primarily associated with the COOH-terminal domain. Specifically, both HL and the LPL/HL chimera were eluted from immobilized heparin by 0.75 M NaCl, whereas 1.1 M NaCl was required to elute LPL and the HL/LPL chimera. Finally, HL is more active than LPL in the hydrolysis of phospholipid substrates. However, the ratio of phospholipase to neutral lipase activity in both chimeric lipases was enhanced by the presence of the heterologous COOH-terminal domain, demonstrating that this domain strongly influences substrate specificity. The NH2-terminal domain thus controls the kinetic parameters of these lipases, whereas the COOH-terminal domain modulates substrate specificity and heparin binding.     

Cell attachment activity of the carboxyl-terminal domain of human thrombospondin expressed in Escherichia coli.

Thrombospondin (TS) is a multidomain, adhesive glycoprotein that associates with cells through multiple cell attachment sites. One of these has been located in or near the globular COOH-terminal region of TS by the monoclonal antibody (mAb) C6.7, which inhibits the attachment of human melanoma cells (G361) to TS. The epitope for C6.7 lies within the last 122 residues of the COOH-terminal domain of TS. This domain is distant from two known cell attachment sites in TS, namely the NH2-terminal heparin-binding domain and the CSVTCG sequences in the type I repeats, but is close to the RGDA sequence, an integrin-dependent cell attachment site. In order to separate the adhesive activity of the TS COOH-terminal domain from that of the RGD sequence, we have expressed the COOH-terminal 212 amino acids (residues 941-1152) of TS in Escherichia coli using the expression vector pRIT2T. The resultant fusion protein is effective in supporting G361 cell attachment even though it lacks the RGD sequence. In addition, the expressed protein inhibits adhesion of G361 cells to intact TS. mAb C6.7 blocks adhesion to the expressed TS COOH-terminal domain whereas GRGDSP and VTCG peptides are not inhibitory. These results show that the TS COOH-terminal domain contains a separate cell adhesion site, defined by mAb C6.7, that is distinct from the other adhesion sites of TS.