Endolyn-78, a membrane glycoprotein present in morphologically diverse components of the endosomal and lysosomal compartments: implications for lysosome biogenesis

A monoclonal antibody (2C5) raised against rat liver lysosomal membranes was used to identify a 78-kD glycoprotein that is present in the membranes of both endosomes and lysosomes and, therefore, is designated endolyn-78. In cultures of rat hepatoma (Fu5C8) and kidney cells (NRK), this glycoprotein could not be labeled with [35S]methionine or with [32P]inorganic phosphate but was easily labeled with [35S]cysteine and [3H]mannose. Pulse-chase experiments and determinations of endoglycosidase H (endo H) sensitivity showed that endolyn-78 is derived from a precursor of Mr 58-62 kD that is processed to the mature form with a t1/2 of 15-30 min. The protein has a 22-kD polypeptide backbone that is detected after a brief pulse in tunicamycin-treated cells. During a chase in the presence of the drug, this is converted into an O-glycosylated product of 46 kD that despite the absence of N-linked oligosaccharides is effectively transferred to lysosomes. This demonstrates that the delivery of endolyn-78 to this organelle is not mediated by the mannose-6-phosphate receptor (MPR). Immunocytochemical experiments showed that endolyn-78 is present in the limiting membranes and the interior membranous structures of morphologically identifiable secondary lysosomes that contain the lysosomal hydrolase beta-glucuronidase, lack the MPR, and could not be labeled with alpha-2-macroglobulin at 18.5 degrees C, a temperature which prevents appearance of endocytosed markers in lysosomes. Endolyn-78 was present at low levels in the plasma membrane and in peripheral tubular endosomes, but was prominent in morphologically diverse components of the endosomal compartment (vacuolar endosomes and various types of multivesicular bodies) which acquired alpha-2-macroglobulin at 18.5 degrees C, and frequently contained substantial levels of the MPR and variable levels of beta-glucuronidase. On the other hand, the MPR was very rarely found in endolyn-containing structures that were not labeled with alpha-2-macroglobulin at the low temperature. Thus, the process of lysosomal maturation appears to involve the progressive delivery of lysosomal enzymes to various types of endosomes that may have already received some of the lysosomal membrane proteins. Although endolyn-78 would be one of the proteins added early to endosomes, other lysosomal membrane proteins may be added only to multivesicular endosomes that represent very advanced stages of maturation.     

Molecular genetic study of human arginase deficiency

We have explored the molecular pathology in 28 individuals homozygous or heterozygous for liver arginase deficiency (hyperargininemia) by a combination of Southern analysis, western blotting, DNA sequencing, and PCR. This cohort represents the majority of arginase-deficient individuals worldwide. Only 2 of 15 homozygous patients on whom red blood cells were available had antigenically cross-reacting material as ascertained by western blot analysis using anti-liver arginase antibody. Southern blots of patient genomic DNAs, cut with a variety of restriction enzymes and probed with a near-full-length (1,450-bp) human liver arginase cDNA clone, detected no gross gene deletions. Loss of a TaqI cleavage site was identified in three individuals: in a homozygous state in a Saudi Arabian patient at one site, at a different site in homozygosity in a German patient, and in heterozygosity in a patient from Australia. The changes in the latter two were localized to exon 8, through amplification of this region by PCR and electrophoretic analysis of the amplified fragment after treatment with TaqI; the precise base changes (Arg291X and Thr290Ser) were confirmed by sequencing. It is interesting that the latter nucleotide variant (Thr290Ser) was found to lie adjacent to the TaqI site rather than within it, though whether such a conservative amino acid substitution represents a true pathologic mutation remains to be determined. We conclude that arginase deficiency, though rare, is a heterogeneous disorder at the genotypic level, generally encompassing a variety of point mutations rather than substantial structural gene deletions.     

Perinatal Steroid Treatments Alter Alloparental and Affiliative Behavior in Prairie Voles

This experiment was designed to examine the hypothesis that perinatal manipulation of gonadal or adrenal steroids can alter the subsequent expression of juvenile parental (alloparenting) and affiliative behavior in prairie voles (Microtus ochrogaster). Corticosterone (PRECORT), testosterone (PRE-TP), or oil injections (PRESES) were given on Prenatal Days 12–20 or on Postnatal Days 1–6 (CORT6, TP6, or SES6, respectively). Alloparenting was reduced significantly in females in the CORT6 group and in males in the TP6 group. Sibling affiliative preferences were increased significantly in PRE-TP females and stranger preferences were increased in TP6 and CORT6 females. The results suggest timing is a critical factor determining whether hormones have a facilitative or inhibitory effect on alloparental and affiliative behavior in prairie voles. In this species, corticosterone and testosterone have similar organizational effects on affiliative behavior in females. Alloparental behavior is inhibited by postnatal corticosterone administration in females and by postnatal testosterone administration in males, whereas prenatal steroid administration had no significant effect on alloparenting in either gender.     

Human neurotrophin-3: A one-step peptide mapping method and complete disulfide characterization of the recombinant protein

Human neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family of neurotrophic factors, and the recombinant protein is being developed as a therapeutic for neurodegenerative diseases. The final product purity and lot-to-lot variation are monitored routinely by peptide mapping. However, only the N-terminal region of NT-3 was susceptible to proteolysis under native conditions. Complete digestion required that the protein be chemically modified by reduction and S-alkylation prior to proteolysis. Complete proteolytic degradation of the protein was achieved simply by an intial denaturation of NT-3 in 6 M guanidinium chloride (pH 6) for 2 hr at 37°C, followed by a tenfold dilution with the digestion buffer (0.1 M Tris-HCl, 1 mM CaCl₂ at pH 7.0) and immediate addition of chymotrypsin at 1% by weight. Direct comparison of the peptide map with an identical aliquot that had been reduced and alkylated also allowed the establishment of the cystine linkages present in NT-3: Cys¹⁴ to Cys⁷⁹, Cys⁵⁷ to Cys¹⁰⁸, and Cys⁶⁷ to Cys¹¹⁰. This disulfide structure is homologous to the NGF family of neurotrophic factors.     

Computational sequence analysis of matrix metalloproteinases

Matrix metalloproteinases (MMP) play a cardinal role in the breakdown of extracellular matrix involved in a variety of biological and pathological processes. Research on MMPs has classified and characterized these enzymes according to their matrix substrate specificity, gene and protein domain structure, and regulation of activity and expression. However, the discovery of new MMPs has introduced a need for a more comprehensive and systematic method of classification and quantitative comparison of known and newly discovered members. This study compiles a sequence alignment, constructs a dendrogram, and calculates physical data and homology percentage assignments in order to obtain further insight into MMP structure-function relationships. Thorough analysis of MMP primary sequence domains, physical data patterns, and statistical analysis of sequence homology yields higher resolution in the similarities and differences that group MMP members.     

Zinc status does not affect aluminum deposition in tissues of rats

To examine whether zinc deficiency would increase the toxicity of dietary aluminum, weanling, male Sprague-Dawley rats were fed purified diets containing either 2 or 30 mg Zn/kg diet, with or without 500 mg Al/kg diet for 28 d. Individually pair-fed rats were fed the 30 mg Zn/kg diet with or without added aluminum to control for inanition secondary to zinc deficiency. Rats fed the 2 μg Zn/kg diet showed evidence of zinc deficiency, including anorexia, growth retardation, and depressed concentrations of zinc in tibias and livers. Zinc deficiency did not significantly increase the concentrations of aluminum in the tibias, livers, kidneys, or regions of the brain examined (cerebrum, cerebellum, midbrain, and hippocampus). Inclusion of aluminum in the diet did not alter aluminum concentrations in the various tissues. Under the conditions of this study, zinc deficiency did not result in greater sensitivity to dietary aluminum exposure.     

Spontaneous methylation of hemoglobin by S-adenosylmethionine by a specific and saturable mechanism

The methyl group from S-adenosylmethionine (AdoMet) is transferred into hemoglobin without any evident involvement of an enzyme. There are multiple sites for incorporation of the methyl group into hemoglobin, since both and chains are methylated. The methyl linkages formed in hemoglobin are stable at both alkaline and acidicpH, and the reaction occurs optimally at slightly below neutralpH. Only a small fraction (2%) of hemoglobin tetramers are methylated under the conditions tested. Acid hydrolysis of [³H-methyl]-labeled hemoglobin and determination of phenylisothiocynate derivatives yields N-methyl lysine, which accounts for about one-half of the incorporated [³H-methyl] radioactivity. Other amino acids are methylated as well, with much of the remaining radioactivity being distributed among one or more of the side chains of histidine, cysteine, and arginine. Methyl group transfer to hemoglobin from AdoMet is slow and inefficient (k _cat/K _m5×10^–2), but the reaction velocity tends toward a plateau with increasing AdoMet concentration in a manner suggesting that saturable binding of AdoMet onto hemoglobin is involved in methyl transfer. The velocity of hemoglobin methylation is inhibited by S-adenosylhomocysteine, the known end-product inhibitor of methyltransferases, a further indication that methyl group transfer involves binding and catalysis by a specific site (or sites) in the hemoglobin molecule. These observations may help to explain the known existence of methylated hemoglobins in erythrocyte.