The role of site-specific N-glycosylation in secretion of soluble forms of rabies virus glycoprotein

Rabies virus glycoprotein is important in the biology and pathogenesis of neurotropic rabies virus infection. This transmembrane glycoprotein is the only viral protein on the surface of virus particles, is the viral attachment protein that facilitates virus uptake by the infected cell, and is the target of the host humoral immune response to infection. The extracellular domain of this glycoprotein has N- glycosylation sequons at Asn37, Asn247, and Asn319. Appropriate glycosylation of these sequons is important in the expression of the glycoprotein. Soluble forms of rabies virus glycoprotein were constructed by insertion of a stop codon just external to the transmembrane domain. Using site-directed mutagenesis and expression in transfected eukaryotic cells, it was possible to compare the effects of site-specific glycosylation on the cell-surface expression and secretion of transmembrane and soluble forms, respectively, of the same glycoprotein. These studies yielded the surprising finding that although any of the three sequons permitted cell surface expression of full-length rabies virus glycoprotein, only the N-glycan at Asn319 permitted secretion of soluble rabies virus glycoprotein. Despite its biological and medical importance, it has not yet been possible to determine the crystal structure of the full-length transmembrane form of rabies virus glycoprotein which contains heterogeneous oligosaccharides. The current studies demonstrate that a soluble form of rabies virus glycoprotein containing only one sequon at Asn319 is efficiently secreted in the presence of the N-glycan processing inhibitor 1-deoxymannojirimycin. Thus, it is possible to purify a conformationally relevant form of rabies virus glycoprotein that contains only one N-glycan with a substantial reduction in its microheterogeneity. This form of the glycoprotein may be particularly useful for future studies aimed at elucidating the three-dimensional structure of this important glycoprotein.      

Loss of alveolar membrane diffusing capacity and pulmonary capillary blood volume in pulmonary arterial hypertension

Background

Reduced gas transfer in patients with pulmonary arterial hypertension (PAH) is traditionally attributed to remodeling and progressive loss of pulmonary arterial vasculature that results in decreased capillary blood volume available for gas exchange.

Methods

We tested this hypothesis by determination of lung diffusing capacity (DL) and its components, the alveolar capillary membrane diffusing capacity (D_m) and lung capillary blood volume (V_c) in 28 individuals with PAH in comparison to 41 healthy individuals, and in 19 PAH patients over time. Using single breath simultaneous measure of diffusion of carbon monoxide (DL_CO) and nitric oxide (DL_NO), DL and D_m were respectively determined, and V_c calculated. D_m and V_c were evaluated over time in relation to standard clinical indicators of disease severity, including brain natriuretic peptide (BNP), 6-minute walk distance (6MWD) and right ventricular systolic pressure (RVSP) by echocardiography.

Results

Both DL_CO and DL_NO were reduced in PAH as compared to controls and the lower DL in PAH was due to loss of both D_m and V_c (all p < 0.01). While DL_CO of PAH patients did not change over time, DL_NO decreased by 24 ml/min/mmHg/year (p = 0.01). Consequently, D_m decreased and V_c tended to increase over time, which led to deterioration of the D_m/V_c ratio, a measure of alveolar-capillary membrane functional efficiency without changes in clinical markers.

Conclusions

The findings indicate that lower than normal gas transfer in PAH is due to loss of both D_m and V_c, but that deterioration of D_m/V_c over time is related to worsening membrane diffusion.     

Sequence variation in ZFX introns in human populations

DNA variation in human populations was studied by examining the last intron of the ZFX gene (about 1, 151 bp) with a worldwide sample of 29 individuals. Only one polymorphic site was found, which is located in an Alu sequence. This polymorphism is present at an intermediate frequency in all populations studied, and could be a shared polymorphism or due to migration among populations in Asia, Europe, and Africa. The nucleotide diversity is 0.04%, supporting the view that the level of nucleotide variation in nuclear DNA is very low in humans. From the sequence data, the age (T) of the most recent common ancestor of the sampled sequences is estimated: the mode of T is about 306,000 years, and the 95% confidence interval of T is 162,000-952,000 years. This mode estimate is considerably older than the estimates from Y- linked sequences.      

Contrasting levels of DNA polymorphism at the autosomal and X-linked visual color pigment loci in humans and squirrel monkeys

The X-linked color pigment (opsin) locus is known to be highly polymorphic in the squirrel monkey and other New World monkeys. To see whether this is also the case for the autosomal (blue) opsin locus, we obtained 32 squirrel monkey and 30 human blue opsin gene sequences. No amino acid polymorphism was found in either the squirrel monkey sample or the human sample, contrary to the situation at the X-linked opsin locus. This sharp contrast in the level of polymorphism might be due to differences in gene expression between the autosomal and the X-linked loci. At the X-linked locus, heterozygote advantage can occur because, owing to X-inactivation, the two alleles in a heterozygote are expressed in different cone cells, producing two types of cone cell, whereas at the autosomal locus, heterozygote advantage cannot occur because the two alleles in a heterozygote are expressed in the same cone cells, producing only one type of cone cell (i.e., phenotypically a homozygote). From the sequence data, the levels of nucleotide diversity (pi, i.e., the number of nucleotide differences per site) are estimated: for the human sample, pi = 0.00% per nondegenerate site, 0.00% per twofold degenerate site, and 0.04% per fourfold degenerate site in the coding regions and 0.01% per site in intron 4; for the squirrel monkey sample, pi = 0.00% per nondegenerate site, 0.00% per twofold degenerate site, and 0.15% per fourfold degenerate site in the coding regions and 0.17% per site in intron 4. The blue opsin genes from the common and pygmy chimpanzees, the gorilla, the capuchin, and the howler monkey were also sequenced. Features critical to the function of the opsin are well conserved in all known mammalian sequences. However, the interhelical loops are, on average, actually more conservative than the transmembrane helical regions. In addition, these sequence data and those from some other genes indicate that the common and pygmy chimpanzees are not closely related, their divergence data being from one third to one half the date of the human-chimpanzee divergence.      

Gene conversion and natural selection in the evolution of X-linked color vision genes in higher primates

During higher primate evolution, gene conversion seems to have occurred often between the red and green photo-pigment genes, which are tandemly linked on the X chromosome. To understand this phenomenon better, intron 4 sequences of the red and green pigment genes of a male human (an Asian Indian), a male chimpanzee, and a male baboon were amplified by PCR and sequenced. The data show that the intron 4 sequences between the two genes have been strongly or completely homogenized in the three species studied. Apparently recent gene conversion events have occurred in introns 4 of the red and green pigment genes in humans and chimpanzees. Two or more conversion events may have occurred at different times in introns 4 of the two pigment genes in baboons. The divergence between the two genes is significantly lower in intron 4 than in exons 4 and 5 in each species, contrary to the usual situation that introns evolve faster than exons. It is most likely that strong natural selection for maintaining the distinct functions of exons 4 and 5 of the red and green pigment genes has acted against sequence homogenization of these exons.      

The rate of synonymous substitution in enterobacterial genes is inversely related to codon usage bias

Genes sequences from Escherichia coli, Salmonella typhimurium, and other members of the Enterobacteriaceae show a negative correlation between the degree of synonymous-codon usage bias and the rate of nucleotide substitution at synonymous sites. In particular, very highly expressed genes have very biased codon usage and accumulate synonymous substitutions very slowly. In contrast, there is little correlation between the degree of codon bias and the rate of protein evolution. It is concluded that both the rate of synonymous substitution and the degree of codon usage bias largely reflect the intensity of selection at the translational level. Because of the high variability among genes in rates of synonymous substitution, separate molecular clocks of synonymous substitution might be required for different genes.      

Intronic gene conversion in the evolution of human X-linked color vision genes

Human red and green visual pigment genes are X-linked duplicate genes. To study their evolutionary history, introns 2 and 4 (1,987 and 1,552 bp, respectively) of human red and green pigment genes were sequenced. Surprisingly, we found that intron 4 sequences of these two genes are identical and that the intron 2 sequences differ by only 0.3%. The low divergences are unexpected because the duplication event producing the two genes is believed to have occurred before the separation of the human and Old World monkey (OWM) lineages. Indeed, the divergences in the two introns are significantly lower than both the synonymous divergence (3.2% +/- 1.1%) and the nonsynonymous divergence (2.0% +/- 0.5%) in the coding sequences (exons 1-6). A comparison of partial sequences of exons 4 and 5 of human and OWM red and green pigment genes supports the hypothesis that the gene duplication occurred before the human-OWM split. In conclusion, the high similarities in the two intron sequences might be due to very recent gene conversion, probably during evolution of the human lineage.      

Effects of sex and dietary phosphorus level on the apparent metabolizable energy and nutrient digestibility in broiler chickens

The influence of sex of broilers and dietary phosphorus (P) level on energy utilization and apparent ileal digestibility of N, P and phytate-P were investigated. The AME_N was determined using 3- and 6-week old broilers, while the apparent ileal digestibility were determined only with 6-week old birds. Sex of broilers had no effect on the AME_N values determined during week 3. During week 6, the AME_N values for male broilers were higher (P?0.01) than those for the females. An interaction (P?0.05) between sex and dietary P level was also observed. AME_N values determined with male broilers were lower in the adequate-P diet compared to those in the low-P diet, whereas no effect of P level was observed in females. Female broilers tended (P?0.10) to have a higher ileal N digestibility than the males, but a significant (P?0.01) sex ×?P level interaction was observed. Males receiving the adequate-P diet had a lower N digestibility than those receiving the low-P diet, whereas the opposite was true in the females. Ileal phytate P degradation in males was higher than in females (0.282 vs. 0.234), but the differences were not significant. Increasing dietary P level increased ileal P digestibility in both the males and females, but the improvements were greater in the females, as indicated by a significant sex × P level interaction.