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Myeloid elf-1-like factor (MEF) or Elf4, which is a member of the ETS transcription factor family, up-regulates the basal expression of lysozyme gene in epithelial cells and is constitutively localized in the nucleus. The mammalian cell nucleus is organized into distinct nuclear domains or compartments that are essential for diverse physiological processes. Promyelocytic leukemia (PML) nuclear body or nuclear domain 10 is one of the nuclear domains and is involved in tumor suppression and regulation of transcription. Here, we investigate the role of PML nuclear body in MEF transactivation. We show that PML, but not Sp100, induced the accumulation of MEF in PML nuclear bodies and that MEF and PML physically interacted. This interaction stimulated MEF transcriptional activity, resulting in the up-regulation of endogenous lysozyme expression. Amino acids 348-517 of MEF were required for the accumulation of MEF in PML nuclear bodies and up-regulation of lysozyme transcription, which is enhanced by PML. Moreover, the C-terminal region of MEF spanning amino acids 477-517 was the putative region required for interaction between MEF and PML as determined with the use of the mammalian two-hybrid system. In addition, heat-shock treatment induced the accumulation of MEF in endogenous PML nuclear bodies and enhanced MEF transactivation of lysozyme gene. Thus, the recruitment of MEF to PML nuclear bodies may partly regulate lysozyme transcription in epithelial cells.

To examine the effect of a conformational constraint introduced into the Arg-Gly-Asp (RGD) sequence on cell adhesion activity, we constructed a mutant protein by inserting an RGD-containing sequence flanked by two Cys residues between Val74 and Asn75 of human lysozyme. The CRGDSC-inserted lysozyme was expressed in yeast, purified, and designated as Cys-RGD4. Using baby hamster kidney cells, Cys-RGD4 was shown to possess even higher cell adhesion activity than that of the RGDS-inserted lysozyme, RGD4. The Cys-RGD4 protein was co-crystallized with a lysozyme inhibitor, tri-N-acetylchitotriose, and the three-dimensional structure was determined at 1.6-A resolution by x-ray crystallography. In contrast to RGD4, the inserted RGD-containing region of Cys-RGD4 was well defined. The structural analysis revealed that the two inserted Cys residues form a new disulfide bond in Cys-RGD4, as expected, and that the RGD region assumes a type II' beta-turn conformation of Gly-Asp with a hydrogen bond between the C = O of Arg and the H-N of Ser. In addition, it was confirmed that two more hydrogen bonds are present in the RGD region of the Cys-RGD4 lysozyme. These results suggest that the conformation of the RGD-containing region is rigid and stable in the Cys-RGD4 molecule and that the type II' beta-turn structure of RGD is essential for binding to integrins with high affinity.

The mechanism of the enhanced bactericidal action to Escherichia coli of the lysozyme having a hydrophobic pentapeptide (Phe-Phe-Val-Ala-Pro) at its C terminus was investigated. The modified lysozyme, hydrophobic pentapeptide-fused lysozyme (HLz), was secreted in the culture medium from yeast harboring the expression plasmid, in which a synthetic DNA fragment encoding a hydrophobic pentapeptide was introduced to the 3'-end of the coding region of the lysozyme cDNA. Although CD analysis showed that HLZ was considerably different from wild-type lysozyme (WLz) in the secondary and tertiary structures, it retained 76% of the lytic activity of WLz. When E. coli cells were exposed to the WLz or HLz, the survival cells were significantly reduced only in the case of HLz. Periplasmic proteins from the HLz-treated cells were released to an extent similar to that from the WLz-treated cells, indicating that HLz has nearly the same action as WLz with respect to the disruption of the outer membrane and peptidoglycan. Experiments with E. coli phospholipid liposomes revealed that HLz dissipated the valinomycin-induced transmembrane electrochemical potential, but WLz did not. These results suggest that the enhanced bactericidal action of HLz to E. coli is due to disruption of the electrochemical potential of the inner membrane in cooperation with the inherent function of the lysozyme to the outer membrane and peptidoglycan.

Chinese hamster ovary cells transfected with human lysozyme cDNA encoding Asn instead of Gly22 synthesize a mutant lysozyme, [Asn22]lysozyme, with about 60% of the molecules bearing carbohydrate. This carbohydrate is predominantly of the complex type and contains a varied number of lactosamine repeats. In this study we show that the glycosylation of [Asn22] lysozyme fused to human cathepsin D is altered relative to [Asn22]lysozyme alone. The fusion protein is synthesized as a 66-kDa precursor that is cleaved to enzymatically active and antigenically positive cathepsin D and lysozyme. As compared with [Asn22]lysozyme the lysozyme moiety of the fusion protein shows an increased N-glycosylation and a decreased synthesis of lactosamine repeats. Cleavage of the precursor with cathepsin L has revealed that the lysozyme portion of the secreted fusion protein bears a complex type carbohydrate. The intracellularly released lysozyme portion of the fusion protein contains trimmed oligosaccharides. In the presence of NH4Cl the lysosomal targeting of the fusion protein is inhibited. The secreted protein is then enriched in molecules bearing phosphorylated high mannose oligosaccharides in their lysozyme moiety. Our results indicate that carbohydrate processing in [Asn22]lysozyme, including the synthesis of mannose 6-phosphate residues and of lactosamine repeats, is altered by the attached cathepsin D. The phosphorylation of the carbohydrate on the lysozyme portion results in a very efficient lysosomal targeting of the concerned fusion protein molecules.

The large molecular size of N-glycosylated lysozyme with a polymannose chain was predominantly expressed in the yeast carrying the lysozyme expression plasmid in 9-fold greater secretion compared with the wild type. Complementary DNA encoding hen egg white lysozyme was subjected to site-directed mutagenesis to obtain the Asn-X-Ser/Thr sequence that is the signal for asparagine-linked (N-linked) glycosylation. At positions 49, 67, 70, and 103, the signal for N-linked glycosylation was created. Only the mutant lysozyme whose glycine 49 was substituted with asparagine was expressed in the two types of glycosylated forms, a small oligomannose chain (Man18GlcNAc2)-linked form and a large polymannose chain (Man310GlcNAc2)-linked form, whereas other mutants were not glycosylated. The secreted amount of polymannosyl lysozyme was much higher than that of the oligomannosyl lysozyme. Both types of glycosylated lysozymes were susceptible to endo-beta-N-acetylglucosaminidase cleavage of their carbohydrate chains. The average molecular masses of oligomannosyl and polymannosyl lysozymes were 18 and 71 kDa, respectively. The length of the polymannose chain was found to be 200-350 residues/molecule of lysozyme according to the estimation of the molecular mass distribution by low angle laser light scattering measurements. The protein conformation estimated by CD analysis was completely conserved in these glycosylated lysozymes. The enzymatic activities of oligomannosyl and polymannosyl lysozymes were 100 and 91%, respectively, of wild-type protein when glycol chitin was used as a substrate. In addition, the polymannosyl lysozyme revealed remarkable heat stability in that no coagulation was observed under conditions in which the wild-type lysozyme coagulated. Thus, this novel glycoprotein can be used as a reporter in studies of the processing and sorting of glycoproteins and as a model of the expression of foreign genes in yeast for the construction of stable enzymes.

Complementary DNA encoding human lysozyme was subjected to oligonucleotide-directed mutagenesis. At one of three selected positions, amino acid residues 22, 68, or 118, the signal for N-linked glycosylation was created. The mutant DNAs were inserted into a eucaryotic vector and transfected into cultured hamster cells. The three mutant cDNAs directed synthesis of lysozyme mutants, which were named LI, LII, and LIII. The mutant lysozymes LI and LII comprised mixtures of glycosylated and nonglycosylated forms. The glycosylated and nonglycosylated forms of mutant LI were found to have an enzymatic activity similar to normal human milk lysozyme. The usage of the glycosylation sites in the mutants was similar in Chinese hamster ovary (CHO) and baby hamster kidney cells. Approximately two of every three molecules in mutant LI, approximately one of every eight molecules in mutant LII, and practically no molecules in mutant LIII became glycosylated. In CHO cells, the processing of the oligosaccharide side chains yielded several larger products than in baby hamster kidney cells. This size variability of glycosylated lysozyme from CHO cells may be explained by the presence of biantennary and triantennary endo-beta-N-acetylglucosaminidase H-resistant oligosaccharides with N-acetyllactosamine repeats of variable length and by the presence of hybrid oligosaccharides, as suggested by affinity to several lectins and sensitivity to endo-beta-galactosidase. In both cell types, the majority of the glycosylated forms were secreted and thus behaved similarly to nonglycosylated lysozyme. A small proportion of mutant LI lysozyme remained associated with the cells. The retained lysozyme was recruited predominantly from the molecules bearing high mannose oligosaccharides. These molecules were targeted to lysosomes, and their carbohydrate was trimmed to an endo-beta-N-acetylglucosaminidase H-resistant form. Owing to the small size of mutant LI lysozyme, minor changes in the size of its carbohydrate moiety result in detectable changes in the electrophoretic mobility of the whole glycoprotein. We suggest that this novel glycoprotein could be used as a reporter in studies on processing and segregation of glycoproteins.

A mutant human lysozyme C77A, in which Cys-77 is replaced with Ala, was secreted by Saccharomyces cerevisiae as two proteins (C77A-a and C77A-b) with different specific activities. A peptide fragment from Val93 to Ala108 was obtained from C77A-a by pepsin digestion, and examined by fast atom bombardment mass spectrometry and amino acid analysis. The results showed that glutathione was attached to the thiol group of Cys95 of the fragment through a disulfide linkage. This observation was confirmed by quantitative formation of free glutathionesulfonic acid from C77A-a by performic acid treatment. In contrast, there was no modification in the case of C77A-b. These results indicate that C77A-a contained a mixed disulfide with glutathione attached to cysteine residue 95. In C77A-b, there appears to be a free thiol of Cys95 surrounded by many side chains, which was not modified by iodoacetic acid under native conditions, suggesting that the attachment of glutathione occurs during folding. These findings further suggest that in the oxidation step of disulfide bond formation in human lysozyme secreted by yeast, mixed disulfides are formed with glutathione and that posttranslational modification with glutathione can occur even in a protein secreted by yeast.

Sequential reverse transcriptase, DNA polymerase, and S1 nuclease reactions can be employed to synthesize double-stranded DNA representing messenger RNA. Using reverse transcriptase products made from partially purified lysozyme, ovomucoid, and ovalbumin messengers from hen oviduct, we have characterized the Escherichia coli DNA polymerase I reaction. We have optimized for a high yield of full length second strands under conditions which require only a small amount of mRNA. The effects of several parameters (time, enzyme levels, salt concentration, monovalent cation, and temperature) on the length of products synthesized by DNA polymerase I have been investigated. Each has a significant influence on the proportion of products which are full length. Under our conditions the three reactions are efficient in synthesizing full length duplex DNA from partially purified mRNA fractions or from total poly(A)-containing RNA.

A large library of hen oviduct cDNA-pCR1 recombinant plasmids has been established in Escherichia coli X1776. From this library, ovomucoid cDNA and lysozyme cDNA-bearing plasmids have been identified. One of these plasmids, pMu7, yielded the sequence of the 3'-untranslated region of ovomucoid mRNA.

Sequences are presented for the signal peptides of prelysozymes from 6 species of birds and compared to the known sequence for chicken prelysozyme c. The sequencing was done with synthetic oligonucleotides as primers and oviduct mRNA as the template, obviating the need to clone DNA from these species. Ring-necked pheasant prelysozyme c differs from all other prelysozymes c and pre-alpha-lactalbumins examined by being cleaved in vivo between amino acid residues 17 and 18 instead of between residues 18 and 19. The feature unique to the signal peptide of pheasant prelysozyme c is proline at position 17. Besides showing that proline is acceptable as the carboxyl-terminal amino acid of the signal peptide, our finding implies that it cannot occur as the penultimate amino acid in the signal peptide. This supports the view that unless a polypeptide has the proper secondary structure, signal peptidase will not cleave it, and that this secondary structure is a beta-turn. Another outcome of this comparative study is an estimate that the mean rate of sequence evolution in the prelysozyme signal peptide is 1%/two million years of divergence, similar to that calculated for the insulin signal peptide. Because this rate is a third of the silent substitution rate, it is likely that one out of every three amino acid substitutions is compatible with signal peptide function.

The messenger RNA coding for the egg white proteins ovalbumin, ovomucoid, and lysozyme were isolated by immunoadsorption of polysomes synthesizing these proteins. Monospecific antibodies against ovalbumin, ovomucoid, and lysozyme, raised in rabbits, were reacted with chicken oviduct polysomes. The antibody-polysome complexes were isolated by immunoadsorption onto sheep anti-rabbit antibodies coupled to an insoluble matrix. The specifically bound polysomes were eluted and the mRNA was obtained by poly(U)-Sepharose chromatography. The three specific RNAs were further purified by preparative gel electrophoresis. The purity of the mRNA preparations was demonstrated by analytical gel electrophoresis, the capability to direct the synthesis of specific protein products in a wheat germ cell-free system, and by hybridization to cDNA transcribed from mRNAoa and mRNAomu. Purified mRNAoa was shown to contain less than 0.1% mRNAomu and purified mRNAomu was about 99% pure with respect to mRNAoa. Purified mRNAly was contaminated with mRNAoa to 0.34% and with mRNAomu to 2.9%.

The in vivo incorporation of deuterated amino acids into egg white proteins of Japanese quail is described. Using a synthetic diet, the level of incorporation of selectively deuterated tyrosine, tryptophan, histidine, and phenylalanine into lysozyme was greater than 80% as demonstrated by proton NMR. Load and load-chase experiments using [3H]phenylalanine or [3H]leucine monitored the fast uptake time (t 1/2 = 2 days) and confirmed the high levels of incorporation. The potential of this system for preparation of other proteins for NMR spectroscopy is discussed.

The signal recognition particle (SRP) has been shown to target nascent secretory and membrane proteins to the endoplasmic reticulum. In the wheat germ cell-free system, SRP arrests the elongation of the nascent chains until the translational complex is docked to the endoplasmic reticulum membrane where the interaction between SRP and docking protein causes a release of the nascent chain arrest. For two secretory proteins, arrested peptides of 70 amino acids have been identified (Walter, P., Ibrahimi, I., and Blobel, G. (1981) J. Cell Biol. 91, 545-550; Meyer, D. I., Krause, E., and Dobberstein, B. (1982) Nature 297, 647-650). By using an in vitro coupled transcription-translation system, we have analyzed SRP arrest and the resulting peptides of the two secretory proteins lysozyme and granulocyte-macrophage colony-stimulating factor and the membrane protein invariant chain. SRP arrested the elongation of all three proteins at multiple sites, giving rise to ladders of arrested peptides. The size of the arrested peptides increased with the time of translation, resulting in mostly full-length pre-peptides after about 40 min. This suggests that SRP arrest in transient rather than stable. Upon addition of microsomes, the SRP arrest was released, and all the blocked peptides could be chased into mature proteins or full-length precursors.

Lysozyme mRNA was translated in a reticulocyte lysate with mixtures of radioactive amino acids. The in vitro product isolated by immunoprecipitation was shown by gel electrophoresis, peptide mapping, and sequence analysis to be larger than lysozyme synthesized in vivo. An NH2-terminal extension was completely sequenced by automated Edman degradation; the phenylthiohydantoins from each cycle were separated by high pressure liquid chromatography and quantitated by scintillation spectroscopy. The NH2-terminal sequence of pre-lysozyme is: (formula: see text) where lysine is the NH2 terminus of lysozyme. Sixteen of the eighteen residues in this sequence are hydrophobic and in this regard it resembles the partial sequences recently elucidated for other secretory proteins. The NH2-terminal methionine is donated by initiator Met-tRNAfMet; thus, this sequence represents the primary translation product. This 18-amino acid sequence is cleaved from lysozyme in vivo before the lysozyme molecules are completely synthesized.