Proteolytic Cleavage Product Research Articles

Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein

We have expressed the human immunodeficiency virus type 1 (HIV-1) gag polyprotein (Pr55gag) in bacteria under the control of the T7 phage gene 10 promoter. When the gene encoding the viral protease is included in cis, in the -1 reading frame, the expected proteolytic cleavage products MA and CA are produced. Disruption of the protease-coding sequence prevents proteolytic processing, and full-length polyprotein is produced. Pr55gag, separated from bacterial proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immobilized on nitrocellulose membranes, binds RNA containing sequences from the 5' end of the HIV-1 genome. This binding is tolerant of a wide range of pH and temperature but has distinct salt preferences. Conditions were identified which prevented nonspecific binding of RNA to bacterial proteins but still allowed binding to Pr55gag. Under these conditions, irrelevant RNA probes lacking HIV-1 sequences bound Pr55gag less efficiently. Quantitation of binding to Pr55gag by HIV-1 RNA probes with deletions mutations demonstrated that there are two regions lying within the HIV-1 gag gene which independently promote binding of RNA to Pr55gag.

Cooperative activation of human factor IX by the human extrinsic pathway of blood coagulation

The activation of human coagulation factor IX by human tissue factor.factor VIIa.PCPS.Ca2+ (TF.VIIa.PCPS.Ca2+) and factor Xa.PCPS.Ca2+ enzyme complexes was investigated. Reactions were performed in a highly purified system consisting of isolated human plasma proteins and recombinant human tissue factor with synthetic phospholipid vesicles (PCPS: 75% phosphatidylcholine (PC), 25% phosphatidylserine (PS)). Factor IX activation was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, [3H]factor IX activation peptide assay, colorimetric substrate thiobenzyl benzyloxycarbonyl-L-lysinate (Z-Lys-SBzl) hydrolysis, and specific incorporation of a fluorescent peptidyl chloromethyl ketone. Factor IX activation by the TF.VIIa.PCPS.Ca2+ enzyme complex was observed to proceed through the obligate non-enzymatic intermediate species factor IX alpha. The simultaneous activation of human coagulation factors IX and X by the TF.VIIa.PCPS.Ca2+ enzyme complex were investigated. When factors IX and X were presented to the TF.VIIa complex, at equal concentrations, it was observed that the rate of factor IX activation remained unchanged while the rate of factor X activation slowed by 45%. When the proteolytic cleavage products of this reaction were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was observed that the intermediate species factor IX alpha was generated more rapidly when factor X was present in the reaction mixture. When factor IX was treated with factor Xa.PCPS in the presence of Ca2+, it was observed that factor IX was rapidly converted to factor IX alpha. The activation of factor IX alpha by the TF.VIIa.PCPS.Ca2+ complex was evaluated, and it was observed that factor IX alpha was activated more rapidly by the TF.VIIa.PCPS.Ca2+ complex than was factor IX itself. These data suggest that factors IX and X, when presented to the TF.VIIa.PCPS.Ca2+ enzyme complex, are both rapidly activated and that factor Xa, which is generated in the initial stages of the extrinsic pathway, participates in the first proteolytic step in the activation of factor IX, the generation of factor IX alpha.

Selective secretion of annexin 1, a protein without a signal sequence, by the human prostate gland

Annexins are primarily intracellular proteins as would be predicted from their lack of hydrophobic signal sequences. However, we now report that the human prostate gland selectively secretes high concentrations of annexin 1 (also called lipocortin 1 and p35) and a proteolytic cleavage product, des1-29-annexin 1, into seminal plasma. Secreted annexin 1 had a blocked amino terminus and was structurally indistinguishable from intracellular annexin 1. Although annexin 1 and the structurally related protein, annexin 4, co-localized to many of the same cells of the ductal epithelium of the prostate, annexin 4 was not secreted. Thus, the secretion of annexin 1 appears to involve a highly selective mechanism that does not involve targeting to the endoplasmic reticulum by a hydrophobic signal sequence.

Amino-terminal sequence homology of two chick kidney mitochondrial proteins immunoisolated with monoclonal antibodies to the cytochrome P450 of 25-hydroxyvitamin D3-1α-hydroxylase

We demonstrate the unique capability of monoclonal antibodies for the specific immunodetection and characterization of two antigenic proteins occurring in normal chick kidney mitochondrial extracts. The antigens were adsorbed to cyanogen bromide-activated Sepharose gel coupled to monoclonal antibodies (MAbs) of the IgM class raised against cytochrome P450(1) alpha, which inhibit equally the 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylase catalytic activities (Mandel et al. 1990 J Clin Lab Immunol, in press). The two identified antigenic proteins are polypeptides with apparent molecular weights of 57,000 and 55,000 daltons. The 1 alpha-hydroxylase cytochrome P450 has been shown to have a molecular weight of 57,000 daltons (Mandel et al. 1990 Biochim Biophys Acta 1034:239-246). The optimal antigen:gel ratio for maximal antigen binding as cytochrome P450 heme, which was determined spectrally, was found to be 1.3 nmol cytochrome P450 per g MAb-coupled Sepharose. At this ratio the total binding capacity of the gel was 1 nmol cytochrome P450 per g Sepharose. The two polypeptides were desorbed with 0.1% Emulgen 911 in 25% glycerol at pH 3.0 and separated by SDS-gel electrophoresis. The amino-terminal sequences of the two antigens were determined by automated Edman degradation with an on-line analyzer of PTH derivatives. The sequences in both antigens were 100% homologous. Complete amino acid composition analysis also revealed that their amino acid compositions were highly similar. These findings suggest that the smaller protein may be a proteolytic cleavage product of the 1 alpha-hydroxylase P450 cytochrome and may represent a putative 24-hydroxylase antigen.

Studies on B-lymphocyte Dysfunctions in Severely Burned Patients

We studied in vitro functional parameters of peripheral blood B-lymphocytes from severely burned patients (n = 10; burn injuries ranging from 25 to 72% TBSA). While the number of B-cells remained unchanged, B-cell proliferation induced by Staphylococcus aureus strain Cowan I (SAC) was normal or even enhanced at early and late phases postburn, but showed a marked suppression during the second to fourth week. A similar pattern was observed for the pokeweed mitogen (PWM)- or SAC-stimulated synthesis of immunoglobulin M (IgM), whereas IgG production was decreased over the whole postburn period monitored. Cytokine (interleukin 4)-induced B-cell activation as indicated by the expression of the CD23 surface antigen was impaired throughout the second to fifth week. In parallel, the release of the proteolytic cleavage product sCD23 which represents a B-cell growth and differentiation factor was reduced. Our data provide evidence that activation, proliferation, and differentiation processes of B-lymphocytes are impaired in severely burned patients, which may contribute to their enhanced susceptibility to infection and sepsis.

Structural study of gizzard caldesmon and its interaction with actin. Binding involves residues of actin also recognised by myosin subfragment 1

The interaction between actin and caldesmon that is associated with the inhibition of actomyosin ATPase activity in smooth muscle has been studied using 1H-NMR spectroscopy. Binding studies using the intact molecules were complemented by the use of thrombic cleavage fragments of both turkey and chicken gizzard caldesmon as well as defined peptides of actin, in order to investigate the conformational properties of caldesmon and to localise regions of the primary structures that participate in protein-protein contacts. The binding of caldesmon is shown to involve distinct segments on the N-terminal region (residues 1-44) of actin, as previously observed for the inhibitory component of the thin filament of striated muscle, troponin I [Levine et al. (1988) Eur. J. Biochem. 153, 389-397]. The comparable structural properties of these tissue-specific inhibitors of actomyosin ATPase and the similarities in their mode of interaction at the N-terminal region of actin suggest common aspects to the structural mechanism for thin-filament regulation in smooth and striated muscle. Unlike the inhibitory interaction of troponin I, however, the binding of caldesmon to the N-terminal region of actin directly involves groups within residues 20-41 of actin that are also recognised by myosin subfragment 1. The complementary segment of caldesmon has been localised to a 15-kDa thrombic fragment (residues 483-578) derived from the N-terminal portion of a 35-kDa proteolytic cleavage product from the C-terminal of caldesmon whose interaction with actin is modulated by calmodulin. The results are discussed in relation to the calcium-mediated mechanism for thin-filament regulation in smooth and striated muscle.

Characterization through cDNA cloning of galactoprotein b3 (Gap b3), a cell surface membrane glycoprotein showing enhanced expression on oncogenic transformation. Identification of Gap b3 as a member of the integrin superfamily.

Galactoprotein b (Gap b) is a group of glycoproteins of fibroblast cell membranes showing enhanced expression in association with oncogenic transformation (Carter, W.G., and Hakomori, S. (1977) Biochem. Biophys. Res. Commun. 76, 299-308). We purified Gap b3 (one of the Gap b group) to homogeneity from hamster fibroblast NIL cells transformed with polyoma virus (NILpy). Partial amino acid sequence was determined, and the cDNA clones encoding this protein were isolated by oligodeoxynucleotide hybridization. The 5.0-kilobase pair cDNA encodes a protein of 1051 amino acid residues containing a signal peptide (32 residues), a large extracellular domain (959 residues), a transmembrane domain (28 residues), and a short cytoplasmic domain (32 residues). The overall structure of Gap b3 is similar to that of the alpha subunit of so-called integrin receptors in the following respects: (i) presence of metal-binding sequences; (ii) location of a transmembrane domain near C terminus; (iii) matched positions of most of the cysteine residues. This, together with extensive amino acid sequence homology, strongly suggests that Gap b3 is a member of the integrin family. Gap b3 consists of two polypeptide chains (Mr = 110,000 and 30,000), which seem to be proteolytic cleavage products connected by disulfide bonds from a precursor protein. Southern hybridization shows the presence of a single copy gene in mice and humans as well. Northern hybridization analysis shows that the enhanced expression of Gap b3 in transformed cells is due to increased transcription of its message.

Limited proteolysis of the vasoactive intestinal peptide receptor: comparison of its folded structure in the membrane-bound and detergent-solubilized states

Limited proteolysis was used to probe and compare the conformation of the rat lung vasoactive intestinal peptide (VIP) receptor in membrane-bound and detergent-solubilized states. It had been shown previously that the activity of the detergent-solubilized VIP receptor is sensitive to the nature of the detergent used for extraction (Patthi, S., Simerson S. and Velicelebi, G. (1988) J. Biol. Chem., 263, 19363–19369). Receptors that were extracted from the membrane using digitonin retained the ability to bind 125I-VIP, while those solubilized in Triton X-100 displayed little or no detectable activity. In order to correlate the differences observed in the activity of the receptor with its folded state, membrane-bound and detergent-solubilized receptors were covalently labeled with 125I-VIP and subjected to limited proteolysis using trypsin, chymotrypsin or carboxypeptidase Y. Digitonin-solubilized receptors most closely resembled the membrane-bound protein in terms of protease sensitivity and proteolytic cleavage products. By contrast, receptors solubilized in Triton X-100 displayed increased sensitivity to proteases and produced distinctly different proteolytic patterns. Thus, the differences observed in the activities of receptors solubilized in digitonin and those solubilized in Triton X-100 could be correlated with detectable differences in the conformation of the protein in each respective detergent solution. These results suggest that digitonin provides an environment that is more compatible with the native folded state of the receptor, similar to its conformation in the membrane.

Human DNA polymerase α catalytic polypeptide binds ConA and RCA and contains a specific labile site in the N-terminus

The catalytic polypeptide of DNA polymerase alpha is often observed in vitro as a family of phosphopolypeptides predominantly of 180 and 165 kDa derived from a single primary structure. The estimated Mr of this polypeptide deduced from the full-length cDNA is 165 kDa. Immunoblot analysis with polyclonal antibodies against peptides of the N- and C-termini of the deduced primary sequence indicates that the observed family of polypeptides from 180 kDa to lower molecular weight results from proteolytic cleavage from the N-terminus. Antibodies against the N-terminal peptide detect only the 180 kDa species suggesting that this higher molecular weight polypeptide may be the result of posttranslational modification of the 165 kDa primary translation product. The catalytic polypeptide is not only phosphorylated but is also found to react with lectins ConA and RCA. N-terminal sequencing of the isolated catalytic polypeptide from human cells and of the recombinant fusion proteins indicates that the often observed 165 kDa polypeptide is the in vitro proteolytic cleavage product of the modified 180 kDa protein at the specific site between lys123 and lys124 within the sequence -RNVKKLAVTKPNN-.

An immunologically related family of apolipoproteins associated with triacylglycerol storage in the Cruciferae

The major apolipoproteins associated with oil-storage bodies have been isolated from the mature seeds of six different species of the family Cruciferae. The apolipoproteins were all of molecular mass 19–20 kDa. They were highly abundant in mature seed tissue, accounting for up to 20% total seed proteins, and were localized exclusively on the membranes of oil-storage bodies. Antibodies were raised in rabbits and mice against the six purified apolipoproteins. In each case, the antibodies specifically recognized 19–20 kDa polypeptides on immunoblots of total seed proteins from 15 different species of the Cruciferae. The extent of the immunological cross-reactivity among the six purified seed apolipoproteins of the Cruciferae was investigated quantitatively using enzyme-linked immunosorbent assay. Very high levels of cross-reactivity were obtained, in contrast to a complete lack of cross-reactivity observed when the major seed apolipoprotein of a non-crucifer, Glycine max, was assayed. Peptide mapping studies showed that the different crucifer seed apolipoproteins gave rise to similar proteolytic cleavage products following treatment with Staphylococcus aureus V8 protease, Lysobacter enzymogenes Lys-C endoprotease, and trypsin. The patterns of immunogenic proteolytic cleavage products of the different apolipoproteins were also similar. We propose that there is a family of abundant 19–20 kDa apolipoproteins in mature seeds of oil-bearing Cruciferae. These apolipoproteins are all major components of the membranes of oil-storage bodies. The apolipoproteins are therefore very closely related with respect to their structure, function, and immunological properties.

Connective tissue activation XXXIII. Biologically active cleavage products of CTAP-III from human platelets

Evidence for three new isoforms of CTAP-III from human platelets is presented; two NH2-terminal cleavage products were identified, CTAP-III (des 1-13) and CTAP-III (des 1-15). CTAP-III (des 1-13) has a pI of 8.6 and is a relatively stable proteolytic cleavage product that retains the capacity to stimulate [14C]GAG synthesis in human synovial cell cultures. CTAP-III (des 1-15) appears to be an elastase or chymotrypsin cleavage product and identical to NAP-2, an entity thought to have neutrophil activating properties.

Analysis of gag proteins from mouse mammary tumor virus

Structural proteins designated p10gag, p21gag, p8gag, p3gag, p27gag, and p14gag from the C3H strain of mouse mammary tumor virus (MMTV) were purified by reversed-phase high-pressure liquid chromatography. The N- and C-terminal amino acid sequences and amino acid composition of each protein were determined and compared with the amino acids encoded by the proviral DNA sequences for the MMTV gag gene. The results show that each of the purified proteins is a proteolytic cleavage product derived from the predicted primary translational product of the gag gene (Pr77gag) and that their order in Pr77gag is p10-pp21-p8-p3-n-p27-p14 (where n represents 17 predicted residues that were not identified among the purified proteins). Purified p10gag lacks the initiator methionine and has a myristoyl group attached in amide linkage to the N-terminal glycine residue predicted by the second codon of the gag gene. The cleavage products are contiguous in the sequence of Pr77gag, and the C-terminal residue of p14gag is encoded by the last codon of the gag gene. By analogy with other retrovirus, p14gag is the viral nucleocapsid protein, p10gag is the matrix protein, and p27gag is the capsid protein of mature MMTV. Proteolytic cleavage sites in MMTV Pr77gag bear a striking resemblance to cleavage sites in the gag precursors of D-type retroviruses, suggesting that these viral proteases have similar specificities.

Binding of thyroxine to human plasma low density lipoprotein through specific interaction with apolipoprotein B (apoB-100)

Human plasma low density lipoprotein (LDL), which binds 0.2% of plasma T 4, was shown to interact with the hormone through its protein moiety, apolipoprotein B-100. LDL and LDL 2, the major subfraction of LDL, were found to have 3 equivalent binding sites for T 4 with K a = 2.5 × 10 6 M −1. Photoaffinity labeling of LDL with inner ring-labeled [ 125I]T 4, followed by SDS-PAGE or agarose-SDS-PAGE of the labeled products, revealed that apoB-100 and its proteolytic cleavage products, apoB-74 and apoB-26, bound [ 125I]T 4. In the presence of 1 or 10 μM T 4, labeling was decreased in 7 separate experiments by 40–53% or 65–86%, respectively, consistent with a K a of ≈ 10 6 M −1. Binding of T 4 to apoB-100 associated with VLDL was also demonstrated by photoaffinity labeling. The observed thyroid hormone binding property of lipid-complexed apoB-100 and the knowledge that receptors for the apolipoprotein exist in various tissues suggest a possible physiological role in thyroid hormone transport.

Differential biosynthesis and posttranslational processing of vasopressin and oxytocin in rat brain during embryonic and postnatal development

The biosynthesis and posttranslational processing of arginine vasopressin (AVP) and oxytocin (OT) peptides in the developing rat brain and pituitary were studied using antibodies and complementary separation methods that permitted a quantitative radioimmunoassay (RIA) analysis of precursor, intermediate, and completely processed forms of the peptides. Precursor forms of the peptides were first detected in rat brain as early as embryonic day (E) 15 for AVP and E17 for OT. Proteolytic cleavage products of the precursors were detected 1 d later for both peptides. AVP was present in a fully processed (amidated) from immediately (E16) and throughout fetal development. OT was cleaved from its precursor starting on E18 but remained in an intermediate (C-terminal extended) form until E21, when amidated OT was first detected in the pituitary. Hence, Pro-AVP processing in the fetus was immediate and complete, whereas Pro-OT processing in the fetus was much slower and incomplete, resulting in the generation of partially processed, nonamidated stable forms of the peptide (OT-Gly10, OT-Gly10-Lys11, and OT-Gly10-Lys11-Arg12). The presence of OT-Gly10-Lys11-Arg12 as a major, stable intermediate form, indicated that the in vivo pattern of endoproteolytic cleavage occurred principally at the C-terminus of the pair of basic amino acids at the tripeptide spacer sequence (Gly-Lys-Arg) in the precursor. Although both precursors were first expressed nearly simultaneously in the brain, the steady-state levels of the precursors were very different throughout fetal life. From E16-E21, the quantities of AVP precursors and peptides were 5- to 10-fold greater than those of OT, suggesting a much higher level of precursor biosynthesis in the AVP neurons. In addition to these differences in the regulation of biosynthesis and processing, AVP peptides were axonally transported to the pituitary 3 d earlier than OT peptides, and in far greater (20-fold) abundance. The early presence and abundance of amidated AVP in the brain and pituitary suggests a trophic function for this peptide during development.

Immunofluorescent and immunochemical evidence for the expression of cytovillin in the microvilli of a wide range of cultured human cells.

We have previously purified a Mr 75,000 protein, cytovillin, from cultured human choriocarcinoma cells (JEG-3) and shown that this protein was specifically confined to the microvillus membrane of these cells. I have now studied the expression and the subcellular distribution of cytovillin in eighteen normal and transformed human cell lines and strains by using immunoblotting and indirect immunofluorescence microscopy. In all cell types, cytovillin was highly enriched in cell surface protrusions. When cell types were ranked according to their staining intensity, choriocarcinoma was highest, then amniotic epithelial cells, other choriocarcinoma cells and tumor cells, and finally fibroblastoid cells. The latter only gave faint diffuse fluorescence on the plasma membrane and, occasionally, on the microvilli. However, detergent extracts of all cell types could be shown to contain cytovillin by the use of immunoblotting techniques. Metabolic pulse-chase labelling experiments with JEG-3 cells demonstrated synthesis of cytovillin as a single-chain polypeptide. No precursor forms or specific proteolytic cleavage products could be seen either by immunoblotting or immunoprecipitation. The protein was found to be very stable with a biologic half-life of about 25 hours. The pI determined by isoelectric focusing was 6.1. These results were consistent with cytovillin being an integral component of the microvilli and other surface extensions of all human cell types examined.

Many protein products from a few loci: assignment of human salivary proline-rich proteins to specific loci.

Earlier studies of protein polymorphisms led to the description of 13 linked loci thought to encode the human salivary proline-rich proteins (PRPs). However, more recent studies at the DNA level have shown that there are only six genes which encode PRPs. The present study was undertaken in order to reconcile these observations. Nucleotide and decoded amino acid sequences from each of the six genes were compared with the available protein sequence data for PRPs. This analysis allowed assignment of the PmF, PmS and Pe proteins to the PRB1 locus, the G1 protein to the PRB3 locus, the Po protein to the PRB4 locus, the Ps protein to the PRB2 locus, and the CON1 and CON2 proteins to the PRB4 locus. Correlations between insertion/deletion RFLPs and PRP protein phenotypes were observed for the PmF, PmS, Gl and CON2 proteins. Our overall analysis indicates that in many instances several proteins previously considered to be the products of separate loci are actually proteolytic cleavage products of a large precursor specified by one or other of the six genes identified at the DNA level. Our analysis also demonstrates that some of the "null" alleles proposed to occur at 11 of the 13 loci in the earlier genetic studies, are actually productive alleles having alterations at proteolytic cleavage sites within the relevant precursor protein. The absence of cleavage leads to the persistence of longer precursor peptides not resolved electrophoretically, concurrently with an absence of the smaller PRPs seen when cleavage occurs.

Evidence That Calpains and Elastase Do Not Produce the von Willebrand Factor Fragments Present in Normal Plasma and IIA von Willebrand Disease

Recent evidence suggests that proteolysis plays an important role in some forms of inherited and acquired von Willebrand disease (vWD). Because calpains and one or more enzymes released from polymorphonuclear leukocytes are known to proteolyze von Willebrand factor (vWF) in vitro with resultant loss of large multimers similar to that seen in IIA vWD, they have been suggested as being responsible for the proteolysis in vivo. Using monoclonal epitope mapping, we have examined the proteolysis of the vWF subunit by porcine calcium-activated neutral proteases (calpains) and human leukocyte elastase to determine whether they produce the vWF proteolytic cleavage products seen in normal individuals and IIA vWD. Purified vWF was digested with porcine calpains I and II. We found no difference in the size, location, and quantity of the fragments produced by calpain I v calpain II. New fragments were detected of approximately 200, 170, 150, and 125 Kd. There was no evidence for generation of the native 140 and 176 Kd fragments. Some loss of the native fragments was seen, which suggests that they were further cleaved. Epitope mapping of the 170- and 150-Kd calpain-cleaved fragments revealed them to be from different parts of the molecule than the regions from which the native 176- and 140-Kd fragments derived. This was further supported by determination of the amino-terminal sequence of the calpain-cleaved 170- and 150-Kd fragments. Digestion of vWF with human leukocyte elastase produced new fragments at 210/205, 190, 170/165, 145/140, and 130/125 Kd. No generation of native fragments was detected. Monoclonal epitope mapping of the 145/140-Kd elastase-cleaved band proved that it derived from the carboxyl-terminal portion of the vWF molecule, whereas the native 140-Kd fragment is derived from the amino-terminal end. Neither calpains nor human leukocyte elastase produced the proteolyzed fragments present in normal and IIA vWD and, therefore, probably do not cause the loss of large multimers that is seen in that disorder.

Evidence that calpains and elastase do not produce the von Willebrand factor fragments present in normal plasma and IIA von Willebrand disease

Recent evidence suggests that proteolysis plays an important role in some forms of inherited and acquired von Willebrand disease (vWD). Because calpains and one or more enzymes released from polymorphonuclear leukocytes are known to proteolyze von Willebrand factor (vWF) in vitro with resultant loss of large multimers similar to that seen in IIA vWD, they have been suggested as being responsible for the proteolysis in vivo. Using monoclonal epitope mapping, we have examined the proteolysis of the vWF subunit by porcine calcium- activated neutral proteases (calpains) and human leukocyte elastase to determine whether they produce the vWF proteolytic cleavage products seen in normal individuals and IIA vWD. Purified vWF was digested with porcine calpains I and II. We found no difference in the size, location, and quantity of the fragments produced by calpain I v calpain II. New fragments were detected of approximately 200, 170, 150, and 125 Kd. There was no evidence for generation of the native 140 and 176 Kd fragments. Some loss of the native fragments was seen, which suggests that they were further cleaved. Epitope mapping of the 170- and 150-Kd calpain-cleaved fragments revealed them to be from different parts of the molecule than the regions from which the native 176- and 140-Kd fragments derived. This was further supported by determination of the amino-terminal sequence of the calpain-cleaved 170- and 150-Kd fragments. Digestion of vWF with human leukocyte elastase produced new fragments at 210/205, 190, 170/165, 145/140, and 130/125 Kd. No generation of native fragments was detected. Monoclonal epitope mapping of the 145/140-Kd elastase-cleaved band proved that it derived from the carboxyl-terminal portion of the vWF molecule, whereas the native 140- Kd fragment is derived from the amino-terminal end. Neither calpains nor human leukocyte elastase produced the proteolyzed fragments present in normal and IIA vWD and, therefore, probably do not cause the loss of large multimers that is seen in that disorder.

The presence and origin of phosphopeptides in human saliva.

The presence of phosphopeptides in whole saliva (saliva expectorated from the mouth) was demonstrated and their origin was evaluated. Whole saliva contained much larger numbers of small phosphopeptides than are found in the glandular secretions. Most of these originated from the acidic proline-rich proteins (PRPs) in the major salivary glands and were formed, after secretion into the oral cavity, as a result of rapid degradation by proteolytic enzymes from extraglandular sources contained in sediment from whole saliva. Some peptides may have been formed by cleavage of basic PRPs, but other phosphoproteins apparently contributed little to the observed phosphopeptides. Most of the enzymes that produced phosphopeptides are serine proteinases. The gel-electrophoretic band patterns of the phosphopeptides obtained from 26 individuals of various acidic-PRP phenotypes were remarkably similar, demonstrating that the enzymes responsible were generally present in the population surveyed and that similar cleavages occur regardless of the nature of the acidic PRPs. Many of these peptides were N-terminal proteolytic cleavage products of acidic PRPs. The N-terminal phosphorylated region of acidic PRPs contains various biological activities, such as inhibition of hydroxyapatite formation, calcium binding and binding to hydroxyapatite, the major mineral of teeth. The demonstration of these phosphopeptides in the saliva that is in contact with the oral surface may therefore be of biological importance.

The mast cell binding site on human immunoglobulin E.

Antibodies of the immunoglobulin E isotype sensitize mast cells and basophils for antigen-induced mediator release by binding through the Fc portion to a high-affinity receptor (Fc epsilon R1, Ka = 10(9)M-1) on the cell surface causing the clinical manifestations of type I hypersensitivity. As the amino acid sequence of the human epsilon chain is now known, attempts have been made to map the Fc epsilon R1 binding site on IgE to a fragment smaller than Fc epsilon using proteolytic cleavage products, none of which proved to be active. Cleavage between the C epsilon 2 and C epsilon 3 domains released two inactive fragments, suggesting that the junction between these segments could be important in receptor binding. This region is protected against protease digestion in the rat IgE complex with the receptor of rat basophilic leukaemia cells. Here we report the mapping of the mast cell receptor binding site on human IgE to a sequence of 76 amino acids at the C epsilon 2/C epsilon 3 junction. Recombinant peptides containing this sequence inhibit passive sensitization of skin mast cells in vivo and sensitize mast cells to degranulation by anti-IgE in vitro almost as efficiently as a myeloma IgE. Fragments containing the separate domains are inactive. Additional sequences are required for rapid assembly of fragments into disulphide-linked dimers, suggesting that a single chain can form the active site. In a three-dimensional model of the human Fc epsilon, the two identical segments are far apart. Each folds to generate a cleft between the C epsilon 2 and C epsilon 3 domains on the surface of the Fc epsilon. The docking of IgE on to mast cells could take place within this cleft.