Julie Ivory Rushbrook

Human cystatin C forms an inactive dimer during intracellular trafficking in transfected CHO cells

To define the cellular processing of human cystatin C as well as to lay the groundwork for investigating its contribution to Icelandic Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA‐I), we have characterized the trafficking, secretion, and extracellular fate of human cystatin C in transfected Chinese hamster ovary (CHO) cells. It is constitutively secreted with an intracellular half‐life of 72 min. Gel filtration of cell lysates revealed the presence of three cystatin C immunoreactive species; an 11 kDa species corresponding to monomeric cystatin C, a 33 kDa complex that is most likely dimeric cystatin C and immunoreactive material, ≥70 kDa, whose composition is unknown. Intracellular monomeric cystatin C is functionally active as a cysteine protease inhibitor, while the dimer is not. Medium from the transfected CHO cells contained only active monomeric cystatin C indicating that the cystatin C dimer, formed during intracellular trafficking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of brefeldin A, there was a large increase in intracellular monomer suggesting that dimer dissociation occurs later in the secretion pathway, after exiting the ER but prior to release from the cell. Extracellular monomeric cystatin C was found to be internalized into lysosomes where it again dimerized, presumably as a consequence of the low pH of late endosome/lysosomes. As a dimer, cystatin C would be prevented from inhibiting the lysosomal cysteine proteases. These results reveal a novel mechanism, transient dimerization, by which cystatin C is inactivated during the early part of its trafficking through the secretory pathway and then reactivated prior to secretion. Similarly, its uptake by the cell also leads to its redimerization in the lysosomal pathway. J. Cell. Physiol. 173:423–432, 1997.

Cellular processing of the amyloidogenic cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type

An important gap in our understanding of the pathogenesis of the amyloidoses is the identification of the cellular events that lead from synthesis of an amyloid precursor protein to its conversion to the amyloid fiber subunit. We address this question by characterizing the effects of an amyloidogenic mutation on the intracellular processing of its protein product. The protein, a mutant of the cysteine protease inhibitor cystatin C, is the amyloid precursor protein in Hereditary Cerebral Hemorrhage with Amyloidosis--Icelandic type (HCHWA-I). The amyloid fibers are composed of mutant cystatin C (L68Q) that lacks the first 10 amino acids. We have previously shown that processing of wild-type cystatin C entails formation of a transient intracellular dimer that dissociates prior to secretion, such that extracellular cystatin C is monomeric. We report here that the cystatin C mutation engenders several alterations in its intracellular trafficking. It forms a stable intracellular dimer that is partially retained in the endoplasmic reticulum and degraded. The bulk of mutant cystatin C that is secreted does not dissociate and is secreted as an inactive dimer. Thus, formation of the stable mutant cystatin C dimer is an early event in the pathogenesis of this disease.

Complexity of myosin species in the avian posterior latissimus dorsi muscle

SummaryThe myosin content of the avian posterior latissimus dorsi muscle, a small fast-twitch muscle similar in fibre type to the much-studied pectoralis major muscle (type IIB), has been explored using high resolution chromatography of the proteolytic fragment known as subfragment-1 and of the products of its limited tryptic digestion, followed by N-terminal sequencing of selected peptides. The complexity of species found greatly exceeds that anticipated from the fibre-type homogeneity of the muscle and from previous studies (Bandmanet al., Cell29 (1982) 645–50; Loweyet al., J. Musc. Res. Cell Motility4 (1983) 695–716; Crow & StockdaleDev. Biol.118 (1986) 333–42). A minimum of four heavy chain species were identified. One form, approximately 40% of the heavy chain complement, appears to be identical to the well-characterized type IIB isoform of the pectoralis major muscle. The remaining species differ from the pectoralis major form in primary sequence. None is identical to the post-hatch isoform of the pectoralis major muscle.

Protein and mRNA analysis of myosin heavy chains in the developing avian pectoralis major muscle.

While the existence of post-hatch and adult myosin heavy chain isoforms in the large, avian type IIB pectoralis major muscle has been clearly established, the number and nature of fast myosin heavy chains during in ovo development and the peri-hatch period have not been resolved. In the present study, developmental fast heavy chain proteins purified by high resolution anion-exchange have been characterized by sequence analysis of a unique CNBr peptide and by complementary mRNA analysis. The four proteins present at 15/16 days in ovo are shown to differ uniquely in primary structure. They correlate with heavy chains II, IV, VI and VII, characterized recently as major or minor species in adult fast muscles using similar methods. These four heavy chains are expressed in a time-dependent fashion from 8 to 16 days in ovo. At the mRNA level, heavy chain VI predominates until 12 days in ovo. Heavy chain IV mRNA is upregulated dramatically at 16 days in ovo preparatory to its proteins predominance in the peri-hatch period. Heavy chains II, IV and V (the post-hatch isoform which replaces heavy chain IV) have major roles in adult fast muscles.

Immunoaffinity purification and dose-response of cholinergic neuronal differentiation factor

A glycoprotein from heart cell-conditioned medium, cholinergic neuronal differentiation factor (CDF), causes a transition from noradrenergic to cholinergic phenotype in cultured rat sympathetic neurons. Although the transition has been known to occur in a dose-dependent manner and CDF has been purified, the examination of a complete dose-response of neurons to CDF has not been possible because sufficient quantities of pure CDF have not been available. A complete dose-response curve is essential for evaluating the biological response of the neurons, for assessing the physiological role of CDF and for understanding the mechanism of action of CDF. We report here an immunoaffinity-purification procedure for CDF with a 73.1% recovery using antibodies raised against a synthetic peptide homologous with the N-terminal region of CDF. This method produced pure CDF in quantities sufficient for examination of the full dose-response range of the neurons. Our main findings are the following. The dose-responses of acetylcholine and catecholamine metabolisms to CDF are different, although the same molecule affects both transmitters. While the half-maximal concentrations for acetylcholine induction (0.20 nM) and for catecholamine suppression (0.28 nM) are similar, the response of catecholamine metabolism begins slowly and saturates at a CDF concentration (5-20 nM) considerably higher than that of acetylcholine (0.6 nM). This may indicate that CDF affects multiple processes in catecholamine metabolism.

Developmental myosin heavy chain progression in avian type IIB muscle fibres.

SummaryMyosin heavy chain species were investigated during development in avian pectoralis major muscle (type IIB fibres) by high resolution anion-exchange chromatography of the myosin head region, subfragment-1. At 15 daysin ovo four distinct fast-type heavy chain species, I, II, III and IV, in order of elution, were identified. By 19 daysin ovo, form IV had become predominant and remained the major species through 3-days post-hatch. This form has been named theperihatch form. Between 3 and 5 days post-hatch, a second massive change occurred such that by 5 days post-hatch a new species, V, apparent at 19 daysin ovo in small amounts, dominated and at 8 days post-hatch was the only heavy chain species present. Form V, which corresponds to that previously identified as theposthatch form, continued as the major species through 20 days post-hatch and was replaced slowly by the adult form. N-terminal sequencing of CNBr peptides from three subfragment-1 heavy chain species, the peri-hatch (form IV), the post-hatch (form V) and adult, revealed differences in amino acid sequence consistent with the three being products of different genes. These results confirm and extend recent reports of complexity in fast heavy chain expression prior to hatching in the chicken (Hofmannet al., 1988; Van Horn & Crow, 1989).

Characterization of the myosin heavy chains of avian adult fast muscles at the protein and mRNA levels

High resolution anion-exchange chromatography of myosin subfragment-1 in avian fast muscles revealed five fast heavy chains (I--V) expressed in muscle-specific patterns. Sequence analysis of a unique peptide established that the proteins differed in primary structure and suggested correlation with heavy chain genes identified independently by Robbins and coworkers. The identities of the isoforms and their expression patterns were confirmed at the mRNA level by a reverse- transcription, 5′-anchored PCR procedure. The fast white pectoralis major muscle possessed heavy chain I, the posterior latissimus dorsi muscle, of similar fibre type, expressed heavy chains I, III and IV. The fast red adductor superficialis muscle expressed either, or both, of heavy chains II and IV. The lateral gastocnemius muscle, of mixed fibre type, expressed heavy chains II--V. In general, heavy chains I, III and V appeared to be favoured in fast white fibres, while heavy chains II and IV were characteristic of fast red fibres. These results imply a greater subtlety of fast muscle function than has previously been appreciated