Molecular characterization of nuclear basic protein HPI1, a putative precursor of human sperm protamines HP2 and HP3

The largest intermediate basic protein HPI1 (101 residues) from human sperm chromatin was isolated and characterized. The amino acid composition and sequence analysis of the protein and of tryptic peptides together with peptide mapping of endoproteinases Lys-C and Glu-C hydrolysates showed that the C-terminal region (residues 45-101) of HPI1 is identical to protamine HP2. These structural data strongly suggest that protein HPI1 is a precursor of human sperm protamines HP2 and HP3 (57 and 54 residues, respectively) as well as of two other intermediate basic proteins HPS1 and HPS2 (69 and 66 residues, respectively) sequenced previously.     

Comparison of the amino acid and nucleotide sequences between human and two guinea pig major basic proteins

By means of reverse-phase HPLC, 2 different proteins were obtained from apparently purified pig eosinophil major basic protein (MBP) and these proteins were named GMPB1 and GMBP2. It was revealed that these 2 components of MBP have similar molecular weights and pI values, although the amino acid compositions were slightly different. In the previous study, we cloned and sequenced GMPB1 cDNA. Here we obtained another clone by plaque hybridization using a screening probe synthesized by means of polymerase chain reaction. After sequencing, it became apparent that this clone corresponded to GMBP2. As in the case of GMBP1, the cDNA of GMBP2 encoded pre-proGMBP2 with 3 domains; signal peptide, acidic pro-portion, and mature GMBP2. By comparing the sequences of GMBP1 and GMBP2, it was revealed that the proteins were quite similar to each other. In addition, their sequences also resembled those of human MBP, especially in the basic domain of mature protein; but no such similarity existed in the pro-portion. Although the molecular weights determined by SDS-PAGE of guinea pig and human MBPs were 11,000 and 9,300, respectively, the calculated molecular weights of these 3 MBPs were all 13.8 kDa. The calculated pI values of GMBP1, GMBP2 and human MBP were 11.7, 11.3 and 11.6, respectively. By means of Harr plot analysis, it was revealed that the amino acid sequences, not only in signal peptides but also in the basic domains of mature proteins, were well conserved between guinea pig and human MBPs.     

Identification of amino acid sequences in the polyomavirus capsid proteins that serve as nuclear localization signals

The molecular mechanism participating in the transport of newly synthesized proteins from the cytoplasm to the nucleus in mammalian cells is poorly understood. Recently, the nuclear localization signal sequences (NLS) of many nuclear proteins have been identified, and most have been found to be composed of a highly basic amino acid stretch. A genetic "subtractive" and a biochemical "additive" approach were used in our studies to identify the NLS's of the polyomavirus structural capsid proteins. An NLS was identified at the N-terminus (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) of the major capsid protein VP1 and at the C-terminus (Glu307 -Glu-Asp-Gly-Pro-Glu-Lys-Lys-Lys-Arg-Arg-Leu318) of the VP2/VP3 minor capsid proteins.     

HP1 proteins are essential for a dynamic nuclear response that rescues the function of perturbed heterochromatin in primary human cells

Cellular information is encoded genetically in the DNA nucleotide sequence and epigenetically by the "histone code," DNA methylation, and higher-order packaging of DNA into chromatin. Cells possess intricate mechanisms to sense and repair damage to DNA and the genetic code. However, nothing is known of the mechanisms, if any, that repair and/or compensate for damage to epigenetically encoded information, predicted to result from perturbation of DNA and histone modifications or other changes in chromatin structure. Here we show that primary human cells respond to a variety of small molecules that perturb DNA and histone modifications by recruiting HP1 proteins to sites of altered pericentromeric heterochromatin. This response is essential to maintain the HP1-binding kinetochore protein hMis12 at kinetochores and to suppress catastrophic mitotic defects. Recruitment of HP1 proteins to pericentromeres depends on histone H3.3 variant deposition, mediated by the HIRA histone chaperone. These data indicate that defects in pericentromeric epigenetic heterochromatin modifications initiate a dynamic HP1-dependent response that rescues pericentromeric heterochromatin function and is essential for viable progression through mitosis.     

A reinvestigation of the amino acid sequences of bovine, rabbit, monkey, and human myelin basic proteins

In order to determine whether bovine, rabbit, and monkey myelin basic proteins (BPs) have the sequence Gly-His or His-Gly at positions corresponding to bovine sequence 76-77, we isolated the tryptic peptides encompassing the sequence in question in these proteins and cleaved them into dipeptides with dipeptidyl aminopeptidase I (EC 3.4.14.1). Analysis by gas chromatography/mass spectrometry of the dipeptides released showed that in no case did His follow Gly or Gly precede His. The identification of peptides Ala-Gln and His-Gly (bovine BP) and Ser-His and Gly-Arg (rabbit and monkey BPs) established the His-Gly sequence. A similar sequence analysis of tryptic peptide (80-91) of human BP confirmed the sequence Thr-Gln-Asp-Glu-Asn-Pro (80-85).     

Cuttlefish sperm protamines. 1. Amino acid sequences of two distinct variants

The amino acid sequences of two cuttlefish protamine variants Sp1 and Sp2 have been established from automated sequence analysis and mass spectrometry data. Sp1 (57 residues) and Sp2 (56 residues) have molecular masses of 8410 and 8253 Da, respectively. They are almost identical proteins which differ only by one residue of arginine and the position of two of the serine residues (14 and 37 in Sp1; 13 and 35 in Sp2). With an arginine content of about 77%, cuttlefish protamine is one of the most basic proteins which have ever been characterized and the first typical protamine sequenced in invertebrates. It is closely similar to sperm basic proteins identified in squids but strongly differs from the protamine-like components isolated from the sperm of bivalve molluscs.     

Peripheral membrane proteins of sarcoplasmic and endoplasmic reticulum. Comparison of carboxyl-terminal amino acid sequences

Peripheral endoplasmic reticulum membrane proteins residing in the lumen of the endoplasmic reticulum occupy the same space as other secreted proteins. The presence of a four amino acid salvage or retention signal (KDEL-COOH = Lys-Asp-Glu-Leu-COOH) at the carboxyl-terminal end of peripheral membrane proteins has been shown to represent a signal or an essential part of a signal for their retention within the endoplasmic reticulum membrane. In heart and skeletal muscle, a number of sarcoplasmic reticulum proteins have recently been identified which are peripheral membrane proteins. The high-affinity calcium-binding protein (55 kilodaltons (kDa] appears to conform to the above described mechanisms and contains the KDEL carboxyl-terminal tetrapeptide. Thyroid hormone binding protein is present in the sarcoplasmic reticulum, in addition to its endoplasmic reticulum location, and has a modified but related tetrapeptide sequence (RDEL = Arg-Asp-Glu-Leu), which also probably functions as the retention signal. Calsequestrin and a 53-kDa glycoprotein, two other peripheral membrane proteins residing in the lumen of the sarcoplasmic reticulum, do not contain the KDEL retention signal. The sarcoplasmic reticulum may have developed a unique retention mechanism(s) for these muscle-specific proteins.     

Amino acid sequences that determine the nuclear localization of yeast histone 2B.

Histone-beta-galactosidase protein fusions were used to identify the domain of yeast histone 2B, which targets this protein to the nucleus. Amino acids 28 to 33 in H2B were required for nuclear localization of such fusion proteins and thus constitute a nuclear localization sequence. The amino acid sequence in this region (Gly-29 Lys Lys Arg Ser Lys Ala) is similar to the nuclear location signal in simian virus 40 large T antigen (Pro-126 Lys Lys Lys Arg Lys Val) (D. Kalderon, B.L. Roberts, W.D. Richardson, and A.E. Smith, Cell 39:499-509, 1984). A point mutation changing lysine 31 to methionine abolished nuclear localization of an H2B-beta-galactosidase fusion protein containing amino acids 1 to 33 of H2B. However, an H2B-beta-galactosidase fusion protein containing both this point mutation and the H2A interaction domain of H2B was nuclear localized. These results suggest that H2A and H2B may be cotransported to the nucleus as a heterodimer.     

Multiple cDNA clones encoding nuclear proteins that bind to the tax-dependent enhancer of HTLV-1: all contain a leucine zipper structure and basic amino acid domain.

A trans-activator protein, p40tax, of human T cell leukemia virus type 1 (HTLV-1) activates its own promoter and cellular promoters of IL-2, IL-2 receptor alpha and GM-CSF genes. We isolated three cDNA clones encoding cellular proteins that bind to the p40tax-dependent enhancer of HTLV-1 by screening a lambda gt11 cDNA library of an HTLV-1 infected cell line. All three proteins, TREB5, TREB7 and TREB36, contained a leucine zipper structure and basic amino acid domain, which are conserved in FOS, JUN and CREB, and also had multiple potential phosphorylation sites. The proteins expressed in Escherichia coli bound to the p40tax-dependent enhancer of the 21 bp sequence, but not to an inactive mutant carrying a mutation in the CRE region. In DNase I footprint analysis, all three proteins protected the 21 bp sequences in the LTR; however, the patterns were not identical to each other. TREB7 and TREB36 protected all three repeats of the 21 bp, but TREB5 protected only the second repeat. TREB7 and TREB36 protected the 5' and middle portions of the 21 bp which are essential for p40tax-mediated trans-activation, whereas TREB5 and CREB1 protected a narrower part of the middle region of the second 21 bp repeat containing the CRE consensus sequence. These structural features and DNA binding properties suggest that TREB proteins are members of a CREB protein family and that some of them (i.e., TREB7 and TREB36) may be involved in p40tax-mediated trans-activation.     

Neisseria pili proteins: amino-terminal amino acid sequences and identification of an unusual amino acid.

The amino-terminal amino acid sequences of the pili proteins from four antigenically dissimilar strains of Neisseria gonorrhoeae, from Neisseria meningiditis, and from Escherichia coli were determined. Although antibodies raised to the pili protein from a given strain of gonococcus cross-reacted poorly or not at all with each of the other strains tested, the amino-terminal sequences were all identical. The meningococcal protein sequence was also identical with the gonococcal sequence through 29 residues, and this sequence was highly homologous to the sequence of the pili protein of Moraxella nonliquifaciens determined by other workers. However, the sequence of the pili protein from E. coli showed no similarity to the other sequences. The gonococcal and meningococcal proteins have an unusual amino acid at the amino termini, N-methylphenylalanine. In addition, the first 24 residues of these proteins have only two hydrophilic residues (at positions 2 and 5) with the rest being predominantly aliphatic hydrophobic amino acids. The preservation of this highly unusual sequence among five antigenically dissimilar Neisseria pili proteins implies a role for the amino-terminal structure in pilus function. The amino terminus may be directly or indirectly (through preservation of tertiary structure) important for the pilus function of facilitating attachment of bacteria to human cells.     

Comparison of the amino acid sequences of hen plasma-, yolk-, and white-riboflavin binding proteins

The amino acid sequence of hen egg yolk-riboflavin binding protein (yolk-RBP) was determined by conventional methods. The sequence was identical with that of hen egg white-riboflavin binding protein except that their carboxyltermini were different, that of yolk-RBP lacked 11 or 13 amino acid residues, while hen plasma-RBP had the same C-terminal sequence as white-RBP. This indicated that the C-terminal 11 or 13 amino acid residues in plasma-RBP might be cleaved off during the incorporation from the blood into the oocyte or in the yolk fluid. Yolk-RBP had the same characteristics as white-RBP, such as N-terminal pyroglutamic acid, polymorphism in the amino acid sequence (Lys/Asn) at the fourteenth residue from the N-terminal end, carbohydrate chains attached to both Asn(36) and Asn(147) residues, and phosphate groups bound to some serine residues in the sequence of Ser(185) to Ser(197) as a cluster. These results led us to the conclusion that yolk- and white-RBPs are bio-synthesized from the same gene in the different organs (liver and oviduct). The carbohydrate composition of yolk-RBP was identical to that of plasma-RBP but different from that of white-RBP showing that the processing of the carbohydrate chains in the liver was different from that in the oviduct.     

Proteinases in the study of amino acid sequences in proteins

The proteolytic action of trypsin, chymotrypsin, submaxillary gland proteinases, Lys-C, Staphylococcus aureus st. V8, Armilarria mellea, Mixobacter AL-2 proteinase II, thermolysin and alpha-lytic proteinase is elucidated from the analysis of te data available on the amino acid sequence studies for above 70 proteins. Properties of a series of commercial enzymic preparations and the way of preferential application of proteinases for studying the amino acid sequence are discussed.     

Variations of sequences and amino acid compositions of proteins that sustain their biological functions: An analysis of the cyclophilin family of proteins.

The sequences of the ubiquitous and phylogenetically diversified cyclophilin family of proteins were divided into six groups, namely, vertebrates, invertebrates, other metazoa, plants, fungi, and prokaryotes. These groups of sequences were aligned with the multiple sequence alignment program Clustal-W. The variations of amino acid substitutions and amino acid compositions for these six groups of cyclophilins were calculated using a novel suite of multiple-sequence alignment analysis routines. The cyclophilins from vertebrates can be divided for at least two distinct structural classes that differ from each other by a variable-length amino acid insert within the loop that links alpha-helix II and beta-strand III. A similar structural feature is also present in the other groups of cyclophilins, namely, those from invertebrates, other metazoa, plants, and fungi. The sequences of cyclophilins from fungi and prokaryotes are more diversified than those from vertebrates, and their alterations involve structures other than the amino acid inserts within the loops. Variations of the hydrophobicity and bulkiness of amino acid substitutions of the aligned sequences were calculated for each group of cyclophilins and for the alignment of all the sequences. The variations have clear asymmetry that may signify the need for modification of the physical properties of certain fragments of cyclophilins that are involved in interactions with various cellular components in the evolving environment.