Molecular structure of human protamine P4 (HP4), a minor basic protein of human sperm nuclei

Protamine HP4 is a minor protein which was purified from human sperm nuclei. It was characterized by its amino acid composition, peptide mapping after digestion with highly specific endoproteinases and finally by its amino acid sequence. Protamine HP4 contains high amounts of arginine, cysteine and histidine. The primary structure of the protein was established by sequence analysis of intact protamine and of its fragments. HP4 is a P2-type protamine of 58 residues (Mr 7783) structurally related to human protamines HP2 and HP3 from which it only differs by an amino-terminal extension of one and four residues, respectively. These three protamines exhibit a close structural relationship with mouse protamine mP2. The heterogeneity of protamines in human sperm nuclei is discussed.     

Isolation and amino-acid sequence analysis of human sperm protamines P1 and P2. Occurrence of two forms of protamine P2

The two protamines of human sperm cell nuclei, P1 and P2, were isolated in pure form after extraction with 6M guanidine/5% mercaptoethanol and alkylation with vinyl pyridine by reversed-phase high-performance liquid chromatography. The amino-acid sequence of protamine P1 was determined by analysing the intact protein and the fragments obtained by cyanogen bromide cleavage. Out of the 50 amino-acid residues 24 are arginines and 6 are cysteines. The sequence of protamine P2 was determined by analysing the intact protein and the fragments resulting from cleavage with endoproteinase Lys-C and thermolysin. Protamine P2 was found to occur in two forms which only differ in their N-terminal regions. The form P2' is three amino-acid residues longer at the N-terminus than the form P2'. Out of the 57 amino-acid residues in the longer form 27 are arginines and 5 are cysteines. Human protamine P1 is highly homologous with the protamines isolated from bull, boar, ram and mouse sperm cells, but human protamine P2 shows a novel type of structure, although also here the dominant amino acids are arginine and cysteine.     

Complete amino acid sequence and location of the five disulfide bridges in porcine pancreatic alpha-amylase

Porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrolase EC 3.2.1.1), a single polypeptide chain, contains nine residues of methionine. Eight different fragments resulting from cleavage of this molecule by cyanogen bromide were characterized. The sequences of six of them have previously been reported. Two missing fragments, CN2 (82 residues) and CN3b1 (76 residues) were purified after breaking of the interpeptidic disulfide bridge and their complete sequence as well as that of the previously purified CN1 peptide (102 residues) are reported here. The location of the three disulfide bridges present in these peptides was determined. Ordering of the carboxymethylated cyanogen bromide fragments was carried out by pulse labeling the amylase chain in vivo. The complete sequence of the porcine pancreatic amylase chain (496 residues) and the location of its five disulfide bridges is presented. Comparison with human and mouse pancreatic and salivary alpha-amylases and with rat pancreatic amylase obtained from the corresponding cDNA nucleotidic sequences shows a high degree of homology between mammalian alpha-amylases.     

Rat sperm protamine. Isolation and sequence analysis

Rat protamine was extracted from S-(pyridylethylated) epididymal sperm cell nuclei with dilute hydrochloric acid. The final purification was achieved by reversed-phase high-performance liquid chromatography. The primary structure was determined by N-terminal sequencing of the total S-(pyridylethylated) protein, and of endoproteinase Lys-C- and thermolysin-derived fragments. Rat protamine consists of 50 amino-acid residues. It is a typical type 1 protamine and differs in two and ten positions from the corresponding mouse and rabbit protamine, respectively. Only 26 positions are invariant in all type 1 mammalian protamines.     

Influence of hemin on the conformation of cyanogen bromide-cleaved peptides of apomyoglobin

The complexes of the three BrCN-cleaved fragments of sperm whale apomyoglobin with hemin were studied by circular dichroism (CD). In native myoglobin, the heme is located in the middle fragment; the isolated peptide (residues 56–131), however, produces little extrinsic Cotton effects by the addition of hemin, although about four molecules of hemin are bound to this peptide. In marked contrast, the COOH-terminal peptide (residues 132–153), which binds three hemin molecules, shows strong Cotton effects in the Soret bands and drastically changes its conformation from unordered to highly helical. The Arg-modified or Lys-deaminated peptide no longer undergoes conformational changes by the addition of hemin, suggesting that the two propionic acid groups of one hemin molecule interact with the Arg residue and one of the Lys residues, which stabilizes the induced helical conformation. The NH₂-terminal peptide (residues 1–55) binds one hemin molecules, and the helicity of this fragment is slightly enhanced by the addition of hemin. Both the CD and difference absorption spectra indicate that the mode of interaction between the peptides and hemin are different for the three apomyoglobin fragments.     

Analysis of peptide residues interacting with MHC molecule or T cell receptor. Can a peptide bind in more than one way to the same MHC molecule?

It has generally been assumed implicitly that one can define amino acid residues of a T cell antigenic determinant peptide that interact with the MHC molecule, i.e., residues that form the "agretope." However, if the same peptide can be seen in different conformations or orientations in the same MHC molecule by different T cells, then we would predict that some residues would appear to interact with the TCR of one T cell clone but with the MHC molecule as the peptide is seen by another T cell clone. To test this hypothesis, we synthesized 36 analogue peptides of an immunogenic fragment (P133-146) of sperm whale myoglobin with three different substitutions for each of 12 amino-acid residues and analyzed the role of each residue for I-Ed-binding and for activation of two Th clones, 14.1 and 14.5, specific for the peptide. The two T cell clones showed slightly different fine specificity from each other in that the truncated peptide P136-144 could stimulate 14.5 but not 14.1. The binding activity of nonstimulatory analogues to the I-Ed molecule was measured by functional inhibition analyses using truncated wild-type peptides as stimulators and nonstimulatory analogues as inhibitors. Paradoxical results were obtained that could not be explained by the peptide binding in a single way to the same I-Ed molecule. Some residues appeared to reciprocally reverse their roles for binding to I-Ed vs binding to the TCR when assessed using T-cell clone 14.5 compared to clone 14.1. These results fit the prediction of the above hypothesis and indicate the possibility that the same peptide, P133-146, can bind in more than one way to the same Ia molecule. The T cell clones, 14.1 and 14.5, appear to recognize different P133-146-I-Ed complexes in which the peptide is bound differently. Moreover, a given residue may not have a unique function of always interacting with the MHC molecule or TCR, but may change from one role to the other as it is presented to different T cells.     

Primary structure of rabbit sperm protamine, the first protamine of its type with an aberrant N-terminal

Rabbit protamine was extracted from S-(pyridylethylated) sperm cell nuclei with hydrochloric acid and then isolated by reversed-phase HPLC. The primary structure was determined by amino acid sequence analysis of the total protein and of fragments obtained by digestion with endoproteinase Lys-C and thermolysin. The protamine contains 49 amino acid residues and is clearly homologous with mammalian type 1 protamines, 47% of the positions being invariant. Surprisingly, rabbit protamine possesses an N-terminal valine residue, whereas all mammalian and several non-mammalian protamine sequences of this type start with alanine, the N-terminal region being remarkably conserved during evolution.     

The major protamine from stallion sperm. Isolation and amino-acid sequence

The major stallion protamine was isolated from sperm cell nuclei by extraction with 6M guanidine/5% mercaptoethanol, alkylation with 4-vinylpyridine and subsequent reversed-phase high-performance liquid chromatography. The primary structure of stallion protamine was determined by N-terminal sequencing of the intact protein and of the fragments obtained from thermolysin cleavage of the S-pyridylethylated and from endoproteinase Lys-C cleavage of the S-aminoethylated protein. Stallion protamine consists of 49 amino-acid residues and shows 49% identity with all other sequenced mammalian type 1 protamines.     

Thyroglobulin processing by thyroidal proteases. Major sites of cleavage by cathepsins B, D, and L

The normal provision of thyroid hormones to the body requires their release from the prohormone, thyroglobulin (Tg). Previous work established the importance of cathepsins B, D, and L (formerly designated cysteine proteinase I) to this process but had not defined the points of proteolytic attack for each enzyme. In the present study we labeled rabbit Tg in vivo with sodium 125I and performed limited digestions with cathepsins B, D, and L, purified from human thyroids. The resultant peptide fragments were analyzed by amino-terminal sequencing and located within the Tg molecule by comparison with the cDNA-derived sequences from human Tg. We identified three cleavage points for cathepsin B, corresponding to P'1 residues 532, 795, and 2487; four cleavage points for cathepsin L, corresponding to P'1 residues 2389, 2452, 2490, and 2657; and four cleavage points for cathepsin D, corresponding to P'1 residues 551, 1835, 2468, and 2643. None of the cleavage points was near Tgs known hormonogenic sites, but these peptide fragments contained three of the four major hormonogenic sites in rabbit Tg, suggesting some preference for their early proteolytic processing. Cathespin B alone among the three endopeptidases had some exopeptidase activity toward Tg. The cleavage specificities for each of the endopeptidases resembled those described with other protein substrates. Thus, cathepsin D preferentially cleaved bonds between hydrophobic residues, and cathespin L cleaved bonds with hydrophobic residues at P2 and P3. Although cathepsin Bs specificity was less obvious, it produced a major cleavage between 2 leucine residues. The existence of three endopeptidases cleaving at different sites shows that Tg proteolysis is a complex process, suggests synergism among their enzyme activities, and provides a physiological mechanism for selective hormone release, including its regulation by TSH.     

Amino acid sequence of the N-terminal non-collagenous segment of dermatosparactic sheep procollagen type I.

The non-collagenous N-terminal segment of type I procollagen from dermatosparactic sheep skin was isolated in the form of the peptide Col 1 from a collagenase digest of the protein. The peptide has a blocked N-terminus, which was identified as pyrrolid-2-one-5-carboxylic acid. Appropriate overlapping fragments were prepared from reduced and alkylated peptide Col 1 by cleavage with trypsin at lysine, arginine and S-aminoethyl-cysteine residues and by cleavage with staphylococcal proteinase at glutamate residues. Amino acid sequence analysis of these fragments by Edman degradation and mass spectrometry established the whole sequence of peptide Col 1 except for a peptide junction (7--8) and a single Asx residue (44), and demonstrated that peptide Col 1 consists of 98 amino acid residues. The N-terminal portion of peptide Col 1 (86 residues) shows an irregular distribution of glycine, whereas the C-terminal portion (12 residues) possesses the triplet structure Gly-Xy and is apparently derived from the precursor-specific collagenous domain of procollagen. The central region of the peptide contains ten cysteine residues located between positions 18 and 73 and shows alternating polar and hydrophobic sequence elements. The regions adjacent to the cysteine-rich portion have a hydrophilic nature and are abundant in glutamic acid. The data are consistent with previous physicochemical and immunological evidence that distinct regions at the N- and C-termini of the non-collagenous domain possess a less rigid conformation than does the central portion of the molecule.     

Condensation of DNA by spermatid basic nuclear proteins

Two transition proteins, TP1 and TP2, participate in the repackaging of the spermatid genome early in mammalian spermiogenesis, coincident with the first detectable changes in chromatin condensation. Using an optical trap and a two-channel flow cell to move single DNA molecules into buffer containing protein, we have measured the rates of DNA condensation and decondensation induced by the binding of Syrian hamster transition proteins TP1 and TP2 and protamines P1 and P2. The results show that both transition proteins condense free DNA, with rates similar to those of protamine 1 and 2. DNA molecules condensed with TP1 were significantly less stable than DNA condensed by protamine or by TP2. Experiments conducted with a peptide corresponding to the C-terminal 25 residues of TP2 showed that this domain is responsible for condensing DNA. Experiments conducted with two fragments of TP1 containing arginine and lysine residues demonstrated that DNA binding by TP1 must involve more than these basic sequences. Zinc facilitated the condensation of DNA by P2 but not by TP2. The dissociation rates of TP2 and P2 from DNA were not affected by the addition of zinc.     

Formation of intraprotamine disulfides in vitro.

When mammalian protamine is dissolved in aqueous buffers at neutral or alkaline pH, both ends of the protein fold inward toward the center of the molecule and form disulfide crosslinks that stabilize several different structures. In the absence of reducing agents, these folded forms of protamine may be visualized and quantitated by gel electrophoresis. Using this technique, we have examined the formation of bull protamine disulfides in solution and describe a variety of factors that affect this process. At pH 8, disulfide-stabilized folded forms of protamine appear within minutes after solubilization of the fully reduced protein. Five different monomers are detected by electrophoresis. Each of these monomers is stabilized by at least one disulfide crosslink and migrates with a distinct mobility, ahead of the fully reduced and extended protein. Under certain conditions, dimers of these folded structures crosslinked by interprotamine disulfides are also formed. The appearance of these disulfide-crosslinked forms of protamine is effected by air oxidation, accelerated at alkaline pH, inhibited upon lowering the pH below pH 7 and eliminated by modifying the protein's cysteine residues. Similar intramolecular disulfides are also produced after the protamine molecule binds to DNA. These results suggest that only those cysteines located within the amino- and carboxyterminal ends of the protein appear to participate in forming intramolecular disulfides in vitro.     

Chromatin organization during spermiogenesis in Octopus vulgaris. II: DNA-interacting proteins

In this article we study the proteins responsible for chromatin condensation during spermiogenesis in the cephalopod Octopus vulgaris. The DNA of ripe sperm nuclei in this species is condensed by a set of five different proteins. Four of these proteins are protamines. The main protamine (Po2), a protein of 44 amino acid residues, is extraordinarily simple (composed of only three different amino acid types: arginine (R), serine (S), and glycine (G). It is a basic molecule consisting of 79.5 mol% arginine residues. The rest of the protamines (Po3, Po4, Po5) are smaller molecules (33, 28, and 30 amino acid residues, respectively) that are homologous among themselves and probably with the main Po2 protamine. The ripe sperm nucleus of O. vulgaris also contains a small quantity of a molecule (Po1) that is similar to Po2 protamine. This protein could represent a Po2 protamine-precursor in a very advanced step of its processing. We discuss the characteristics of these proteins, as well as the relation between the complexity of chromatin condensation and the transitions of nuclear proteins during spermiogenesis in O. vulgaris.     

Some structural and functional aspects of the cell adhesion molecule uvomorulin

The cell adhesion molecule uvomorulin (UM) was analysed by comparing antisera produced against the whole molecule (gp123) with antisera made against fragments of UM. Of the proteins recognized by different anti-UM antisera (molecular weights of 123, 102, 92 and 84 kDa), the 102 kDa molecule is not derived from gp123. The 102 kDa molecule is not glycosylated and is also different from gp123 by peptide map analysis. However, rabbit antisera raised against the purified 102 kDa protein interfered with the aggregation of embryonal carcinoma (EC) cells. Also, a monoclonal antibody selected to interfere with EC cell aggregation recognized the 102 kDa molecule as well as gp123. Thus, the functional site of cell adhesion seems not to be mediated by sugar residues. Experimental evidence is provided suggesting that UM is not only involved in the compaction of preimplantation embryos but seems to be an ubiquitous cell adhesion molecule regulating epithelial cell adhesion mechanisms.     

The pigeon cytochrome c-specific T cell response of low responder mice. I. Identification of antigenic determinants on fragment 1 to 65

An examination of the proliferative response to pigeon cytochrome c fragments 1 to 65 and 1 to 80 by T cells from mice that are low responders to the native molecule revealed that some of the strains could respond to antigenic determinants on these fragments. T cell clones derived from B10.A(3R) and B10.A(4R) mice were used to characterize the antigenic determinants on fragment 1 to 65. All of the clones recognized syngeneic A beta:A alpha Ia molecules as their restriction element. Three B10.A(3R) clones and six B10.A(4R) clones recognized fragment 39 to 65. Another four B10.A(4R) clones responded to fragment 1 to 38. By stimulating with a series of cytochrome c fragments from different species, as well as a synthetic peptide, it was possible to localize the antigenic determinant(s) recognized by the B10.A(3R) clones to residues 45 to 58. Each clone showed a unique pattern of responsiveness to the various fragments, suggesting a diversity of T cell receptors specific for the same peptide. One B10.A(3R) clone could be stimulated by many of the 1 to 65 fragments in association with allogeneic B10.SM presenting cells and by tuna fragment 1 to 65 in association with B10.M presenting cells, although the rank order of potency for several of the fragments was different than that observed with syngeneic antigen-presenting cells. In addition, the clone was poorly reactive to a synthetic peptide containing a conservative substitution, serine for threonine, at position 49. The implications of these results for subsite dissection (agretope and epitope) of the antigenic determinant recognized by this clone are discussed.     

Sequences of large T1 ribonuclease-resistant oligoribonucleotides from protamine mRNA: the overall architecture of protamine mRNA.

Limited T1 ribonuclease digestion of the family of protamine mRNA's purified from rainbow trout testis yields several large oligoribonucleotide fragments ranging in size from 12--54 nucleotides in length. Several of these fragments purified by two dimensional gel electrophoresis contain several G residues and must represent nuclease-resistant, base-paired regions of the mRNA. Sequence analysis of these oligonucleotides by the method of Simoncsits, A., Brownlee, G.G., Brown, R.S., Rubin, J.R. and Guilley, H. (1977) Nature 269: 833-836, shows that these oligoribonucleotides arise from the 5'- and 3'-non-coding regions of the mRNA. Comparisons of the sequences of the large RNA fragment with DNA sequences obtained after cloning double-stranded protamine cDNA in the plasmids pBr322 and pmB9 show precise correspondence of a 54 nucleotide RNA fragment with positions 49--100 from the 3'-poly(A) tract and extending to within 5 nucleotides of the termination codon. Two other RNA fragments of 21 and 25 nucleotides in length arise from the 5'-non-coding region of the message and possess an AUG-sequence at their 3'-termini which is the initiation codon. The presence of distinct by homologous sequences in several sets of large RNA fragments is consistent with the presence of several closely related protamine mRNA's.     

Interdomain cleavage of plasma fibronectin by zinc-metalloproteinase from Serratia marcescens

Limited proteolysis of porcine plasma fibronectin by the 56 kDa proteinase (56K proteinase) (EC 3.4.24.4) from Serratia marcescens released six polypeptides: a 27 kDa peptide, the heparin-binding domain which comprises the NH2-terminal end; a 50 kDa peptide, a mid-molecule that mediates binding to gelatin or collagen; a 160 kDa peptide, that contained the heparin-binding domain with cell-spreading activity; and a 140 and a 20 kDa peptide which released from the 160 kDa peptide. Each fragment was purified and characterized by its chemical and biological properties, and it was found that they were respectively different domains. Both the 160 and the 140 kDa peptide contained one cysteine per mole of peptide. The 160 kDa peptides were connected by a 6 kDa peptide, which was present at the COOH-terminal end of the molecule and was biologically inactive. Only 6 kDa peptide contained a disulfide bond and produced 3 kDa peptide after reduction, whereas other fragments did not change with or without reduction on SDS-polyacrylamide gel electrophoresis. NH2-terminal sequence analyses of the released peptides showed that the 56K proteinase cleaved the fibronectin between the Arg-Thr (located at two different sites), Leu-Ser and Gln-Glu bonds. Out of 118 Arg residues, there are nine sequences containing Arg-Thr, and two of them near or at an interdomain location (at Arg 259 and 2239) were cleaved. Out of 124 Leu residues, there are 11 Leu-Ser sequences and only one, at 687, was cleaved. The above fragments with functional domain activity could be aligned according to the previously reported amino-acid sequence of human or bovine plasma fibronectin. The treatment of fibroblast cells by the 56K proteinase resulted in loss of morphological integrity and extracellular matrix.     

Enhanced level of site-specific proteolysis of GAP-43 protein during early stages of brain development

GAP-43 protein of nerve terminals (B-50, F1, F57, pp46, neuromodulin) is thought to be one of key proteins involved in the control of outgrowth of neurites, release of neuromediators, synapse plasticity, etc. GAP-43 is usually considered as a whole protein. Along with the intact protein, nerve cells also contain two large native fragments of GAP-43 deprived of four or of about forty N-terminal amino acid residues (GAP-43-2 and GAP-43-3, respectively). The full-length GAP-43 is predominant in the mature brain. However, the ratio of the full-length protein and its fragments can vary under different physiological conditions. Changes in the GAP-43 proteins (the full-length protein and its fragments) were studied during embryonal and postnatal development of rat brain. The GAP-43 proteins were found to be expressed not later than on the 12-13th day of embryogenesis. Then their contents increased, and, until the 10th day after birth, GAP-43-3 dominated rather than the full-length protein. It is suggested that during this period the activity of a specific protease, which cleaves the N-terminal peptide of about 40 residues from the full-length GAP-43 molecule, is increased. The cleavage occurs in the region responsible for the interaction of GAP-43 with calmodulin. In the full-length molecule, this region is responsible also for the recognition of Ser41 residue by protein kinase C during phosphorylation. Another functionally important region that determines, in particular, the attachment of GAP-43 to the plasma membrane is cleaved from the main part of the molecule together with the N-terminal peptide. Thus, the specific fragmentation of GAP-43 that depends on developmental stage should be considered as a controlled structural rearrangement fundamentally affecting the functions of this protein.     

Molecular orientation of plastocyanin on spinach thylakoid membranes as determined by acetylation of lysine residues

To reveal the molecular orientation of plastocyanin (PC) on spinach thylakoid membranes, the position of Lys residues modified by acetic anhydride was compared between thylakoid-bound PC and the isolated one. Digestion of the isolated PC by a trypsin yielded a peptide map with seven spots prior to the acetylation of the protein; none of the spots appeared after the isolated PC was acetylated. On the other hand, there were two spots on the peptide map of the PC acetylated when it was bound to the thylakoids. Those spots were revealed by their amino acid compositions to correspond to the peptide fragments Phe 82-Lys 95 and Val 96-Asn 99. Thus, the Lys residues 81 and 95 of the thylakoid-bound PC were not acetylated. These results suggest that the PC molecule binds to the thylakoids with a specific region including the Lys's 81 and 95 in contact with the membranes. The Lys's 81 and 95 are located near Tyr 83, which has been thought to be the delivery site of electrons from the Cu2+ center.     

Class II-restricted T-cell clones to a synthetic peptide of influenza virus hemagglutinin differ in their fine specificities and in the ability to respond to virus.

Fifteen T-cell clones were derived from BALB/c or DBA/2 mice immunized with a synthetic peptide corresponding to the C-terminal 24 residues (residues 305 to 328) of the HA1 chain of H3 subtype influenza virus hemagglutinin. All of the clones proliferated when the peptide was presented in association with I-Ed. By using shorter homologs, it was shown that the T-cell response was focused predominantly on the region at the N-terminal end of the peptide encompassed by residues 306 to 319. Individual clones recognizing this region differed in their absolute requirements for residues at the extremities of the site and also in their patterns of efficiency of recognition of shorter homologs. One particular clone defined another site of T-cell recognition within residues 314 to 328. The response of the clones to peptide analogs identified certain residues within the sites that were critical for recognition, with the substitution Gln-311----Ser having a differential effect on clones responding to the N-terminal site. Only one of the clones responded well to influenza virus itself. This clone also required relatively low concentrations of the parent peptide for optimum stimulation and was suppressed by higher concentrations. The data demonstrate striking heterogeneity in the T-cell response even to a short synthetic peptide, with different T-cell clones recognizing slightly different but overlapping areas of the molecule.