System-based proteomic and metabonomic analysis of the Df(16)A+/- mouse identifies potential miR-185 targets and molecular pathway alterations.

Deletions on chromosome 22q11.2 are a strong genetic risk factor for development of schizophrenia and cognitive dysfunction. We employed shotgun liquid chromatography-mass spectrometry (LC-MS) proteomic and metabonomic profiling approaches on prefrontal cortex (PFC) and hippocampal (HPC) tissue from Df(16)A+/- mice, a model of the 22q11.2 deletion syndrome. Proteomic results were compared with previous transcriptomic profiling studies of the same brain regions. The aim was to investigate how the combined effect of the 22q11.2 deletion and the corresponding miRNA dysregulation affects the cell biology at the systems level. The proteomic brain profiling analysis revealed PFC and HPC changes in various molecular pathways associated with chromatin remodelling and RNA transcription, indicative of an epigenetic component of the 22q11.2DS. Further, alterations in glycolysis/gluconeogenesis, mitochondrial function and lipid biosynthesis were identified. Metabonomic profiling substantiated the proteomic findings by identifying changes in 22q11.2 deletion syndrome (22q11.2DS)-related pathways, such as changes in ceramide phosphoethanolamines, sphingomyelin, carnitines, tyrosine derivates and panthothenic acid. The proteomic findings were confirmed using selected reaction monitoring mass spectrometry, validating decreased levels of several proteins encoded on 22q11.2, increased levels of the computationally predicted putative miR-185 targets UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase 110 kDa subunit (OGT1) and kinesin heavy chain isoform 5A and alterations in the non-miR-185 targets serine/threonine-protein phosphatase 2B catalytic subunit gamma isoform, neurofilament light chain and vesicular glutamate transporter 1. Furthermore, alterations in the proteins associated with mammalian target of rapamycin signalling were detected in the PFC and with glutamatergic signalling in the hippocampus. Based on the proteomic and metabonomic findings, we were able to develop a schematic model summarizing the most prominent molecular network findings in the Df(16)A+/- mouse. Interestingly, the implicated pathways can be linked to one of the most consistent and strongest proteomic candidates, (OGT1), which is a predicted miR-185 target. Our results provide novel insights into system-biological mechanisms associated with the 22q11DS, which may be linked to cognitive dysfunction and an increased risk to develop schizophrenia. Further investigation of these pathways could help to identify novel drug targets for the treatment of schizophrenia.

Development of a blood-based molecular biomarker test for identification of schizophrenia before disease onset.

Recent research efforts have progressively shifted towards preventative psychiatry and prognostic identification of individuals before disease onset. We describe the development of a serum biomarker test for the identification of individuals at risk of developing schizophrenia based on multiplex immunoassay profiling analysis of 957 serum samples. First, we conducted a meta-analysis of five independent cohorts of 127 first-onset drug-naive schizophrenia patients and 204 controls. Using least absolute shrinkage and selection operator regression, we identified an optimal panel of 26 biomarkers that best discriminated patients and controls. Next, we successfully validated this biomarker panel using two independent validation cohorts of 93 patients and 88 controls, which yielded an area under the curve (AUC) of 0.97 (0.95-1.00) for schizophrenia detection. Finally, we tested its predictive performance for identifying patients before onset of psychosis using two cohorts of 445 pre-onset or at-risk individuals. The predictive performance achieved by the panel was excellent for identifying USA military personnel (AUC: 0.90 (0.86-0.95)) and help-seeking prodromal individuals (AUC: 0.82 (0.71-0.93)) who developed schizophrenia up to 2 years after baseline sampling. The performance increased further using the latter cohort following the incorporation of CAARMS (Comprehensive Assessment of At-Risk Mental State) positive subscale symptom scores into the model (AUC: 0.90 (0.82-0.98)). The current findings may represent the first successful step towards a test that could address the clinical need for early intervention in psychiatry. Further developments of a combined molecular/symptom-based test will aid clinicians in the identification of vulnerable patients early in the disease process, allowing more effective therapeutic intervention before overt disease onset.

Evidence for disturbed insulin and growth hormone signaling as potential risk factors in the development of schizophrenia.

Molecular abnormalities in metabolic, hormonal and immune pathways are present in peripheral body fluids of a significant subgroup of schizophrenia patients. The authors have tested whether such disturbances also occur in psychiatrically ill and unaffected siblings of schizophrenia patients with the aim of identifying potential contributing factors to disease vulnerability. The subjects were recruited as part of the Genetic Risk and OUtcome of Psychosis (GROUP) study. The authors used multiplexed immunoassays to measure the levels of 184 molecules in serum from 112 schizophrenia patients, 133 siblings and 87 unrelated controls. Consistent with the findings of previous studies, serum from schizophrenia patients contained higher levels of insulin, C-peptide and proinsulin, decreased levels of growth hormone and altered concentrations of molecules involved in inflammation. In addition, significant differences were found in the levels of some of these proteins in siblings diagnosed with mood disorders (n=16) and in unaffected siblings (n=117). Most significantly, the insulin/growth hormone ratio was higher across all groups compared with the controls. Taken together, these findings suggest the presence of a molecular endophenotype involving disruption of insulin and growth factor signaling pathways as an increased risk factor for schizophrenia.

Diabetic db/db mice exhibit central nervous system and peripheral molecular alterations as seen in neurological disorders.

The db/db mouse is a widely used preclinical model in diabetes research. Recent studies have shown that these mice also display aspects of psychosis and depression-like behaviors as seen in some psychiatric disorders. Here, we have performed multiplex immunoassay and liquid chromatography mass spectrometry profiling of the plasma and brain samples from db/db and control mice to identify altered pathways, which could be related to these behavioral abnormalities. This is the first study to carry out profiling of the brain proteome in this model. Plasma from the db/db mice had increased levels of leptin and insulin, decreased levels of peptide YY, glucagon and prolactin and alterations in inflammation-related proteins, compared with control mice. Frontal cortex tissue from the db/db mice showed changes in proteins involved in energy metabolism, cellular structure and neural functioning, and the hippocampus had changes in proteins involved in the same pathways, with additional effects on cellular signalling proteins. The overlap of these findings with effects seen in type 2 diabetes, schizophrenia, major depressive disorder and Alzheimer's disease might contribute to a common endophenotype seen in metabolic and neurological disorders.

Identification of blood-based molecular signatures for prediction of response and relapse in schizophrenia patients.

The current inability of psychiatric medicine to objectively select the most appropriate treatment or to predict imminent relapse are major factors contributing to the severity and clinical burden of schizophrenia. We have previously used multiplexed immunoassays to show that schizophrenia patients have a distinctive molecular signature in serum compared with healthy control subjects. In the present study, we used the same approach to measure biomarkers in a population of 77 schizophrenia patients who were followed up over 25 months with four aims: (1) to identify molecules associated with symptom severity in antipsychotic naive and unmedicated patients, (2) to determine biomarker signatures that could predict response over a 6-week treatment period, (3) to identify molecular panels that could predict the time to relapse in a cross-sectional population of patients in remission and (4) to investigate how the biological relapse signature changed throughout the treatment course. This led to identification of molecular signatures that could predict symptom improvement over the first 6 weeks of treatment as well as predict time to relapse in a subset of 18 patients who experienced recurrence of symptoms. This study provides the groundwork for the development of novel objective clinical tests that can help psychiatrists in the clinical management of schizophrenia.

Identification of proteomic signatures associated with depression and psychotic depression in post-mortem brains from major depression patients.

Major depressive disorder (MDD) is a leading cause of disability worldwide and results tragically in the loss of almost one million lives in Western societies every year. This is due to poor understanding of the disease pathophysiology and lack of empirical medical tests for accurate diagnosis or for guiding antidepressant treatment strategies. Here, we have used shotgun proteomics in the analysis of post-mortem dorsolateral prefrontal cortex brain tissue from 24 MDD patients and 12 matched controls. Brain proteomes were pre-fractionated by gel electrophoresis and further analyzed by shotgun data-independent label-free liquid chromatography-mass spectrometry. This led to identification of distinct proteome fingerprints between MDD and control subjects. Some of these differences were validated by Western blot or selected reaction monitoring mass spectrometry. This included proteins associated with energy metabolism and synaptic function and we also found changes in the histidine triad nucleotide-binding protein 1 (HINT1), which has been implicated recently in regulation of mood and behavior. We also found differential proteome profiles in MDD with (n=11) and without (n=12) psychosis. Interestingly, the psychosis fingerprint showed a marked overlap to changes seen in the brain proteome of schizophrenia patients. These findings suggest that it may be possible to contribute to the disease understanding by distinguishing different subtypes of MDD based on distinct brain proteomic profiles.

Identification of a biological signature for schizophrenia in serum.

Biomarkers are now used in many areas of medicine but are still lacking for psychiatric conditions such as schizophrenia (SCZ). We have used a multiplex molecular profiling approach to measure serum concentrations of 181 proteins and small molecules in 250 first and recent onset SCZ, 35 major depressive disorder (MDD), 32 euthymic bipolar disorder (BPD), 45 Asperger syndrome and 280 control subjects. Preliminary analysis resulted in identification of a signature comprised of 34 analytes in a cohort of closely matched SCZ (n=71) and control (n=59) subjects. Partial least squares discriminant analysis using this signature gave a separation of 60-75% of SCZ subjects from controls across five independent cohorts. The same analysis also gave a separation of ~50% of MDD patients and 10-20% of BPD and Asperger syndrome subjects from controls. These results demonstrate for the first time that a biological signature for SCZ can be identified in blood serum. This study lays the groundwork for development of a diagnostic test that can be used as an aid for distinguishing SCZ subjects from healthy controls and from those affected by related psychiatric illnesses with overlapping symptoms.

Impaired glycolytic response in peripheral blood mononuclear cells of first-onset antipsychotic-naive schizophrenia patients.

Little is known about the biological mechanisms underpinning the pathology of schizophrenia. We have analysed the proteome of stimulated and unstimulated peripheral blood mononuclear cells (PBMCs) from schizophrenia patients and controls as a potential model of altered cellular signaling using liquid-chromatography mass spectrometry proteomic profiling. PBMCs from patients and controls were stimulated for 72 h in vitro using staphylococcal enterotoxin B. In total, 18 differentially expressed proteins between first-onset, antipsychotic-naive patients and controls in the unstimulated and stimulated conditions were identified. Remarkably, eight of these proteins were associated with the glycolytic pathway and patient-control differences were more prominent in stimulated compared with unstimulated PBMCs. None of these proteins were altered in chronically ill antipsychotic-treated patients. Non-linear multivariate statistical analysis showed that small subsets of these proteins could be used as a signal for distinguishing first-onset patients from controls with high precision. Functional analysis of PBMCs did not reveal any difference in the glycolytic rate between patients and controls despite increased levels of lactate and the glucose transporter-1, and decreased levels of the insulin receptor in patients. In addition, subjects showed increased serum levels of insulin, consistent with the idea that some schizophrenia patients are insulin resistant. These results show that schizophrenia patients respond differently to PBMC activation and this is manifested at disease onset and may be modulated by antipsychotic treatment. The glycolytic protein signature associated with this effect could therefore be of diagnostic and prognostic value. Moreover, these results highlight the importance of using cells for functional discovery and show that it may not be sufficient to measure protein expression levels in static states.

Evidence for disease and antipsychotic medication effects in post-mortem brain from schizophrenia patients.

Extensive research has been conducted on post-mortem brain tissue in schizophrenia (SCZ), particularly the dorsolateral prefrontal cortex (DLPFC). However, to what extent the reported changes are due to the disorder itself, and which are the cumulative effects of lifetime medication remains to be determined. In this study, we employed label-free liquid chromatography-mass spectrometry-based proteomic and proton nuclear magnetic resonance-based metabonomic profiling approaches to investigate DLPFC tissue from two cohorts of SCZ patients grouped according to their lifetime antipsychotic dose, together with tissue from bipolar disorder (BPD) subjects, and normal controls (n=10 per group). Both techniques showed profound changes in tissue from low-cumulative-medication SCZ subjects, but few changes in tissue from medium-cumulative-medication subjects. Protein expression changes were validated by Western blot and investigated further in a third group of subjects who were subjected to high-cumulative-medication over the course of their lifetime. Furthermore, key protein expression and metabolite level changes correlated significantly with lifetime antipsychotic dose. This suggests that the detected changes are present before antipsychotic therapy and, moreover, may be normalized with treatment. Overall, our analyses revealed novel protein and metabolite changes in low-cumulative-medication subjects associated with synaptogenesis, neuritic dynamics, presynaptic vesicle cycling, amino acid and glutamine metabolism, and energy buffering systems. Most of these markers were altered specifically in SCZ as determined by analysis of the same brain region from BPD patients.

Sex-specific serum biomarker patterns in adults with Asperger's syndrome.

Autism spectrum conditions have been hypothesized to be an exaggeration of normal male low-empathizing and high-systemizing behaviors. We tested this hypothesis at the molecular level by performing comprehensive multi-analyte profiling of blood serum from adult subjects with Asperger's syndrome (AS) compared with controls. This led to identification of distinct sex-specific biomarker fingerprints for male and female subjects. Males with AS showed altered levels of 24 biomarkers including increased levels of cytokines and other inflammatory molecules. Multivariate statistical classification of males using this panel of 24 biomarkers revealed a marked separation between AS and controls with a sensitivity of 0.86 and specificity of 0.88. Testing this same panel in females did not result in a separation between the AS and control groups. In contrast, AS females showed altered levels of 17 biomarkers including growth factors and hormones such as androgens, growth hormone and insulin-related molecules. Classification of females using this biomarker panel resulted in a separation between AS and controls with sensitivities and specificities of 0.96 and 0.83, respectively, and testing this same panel in the male group did not result in a separation between the AS and control groups. The finding of elevated testosterone in AS females confirmed predictions from the 'extreme male brain' and androgen theories of autism spectrum conditions. We conclude that to understand the etiology and development of autism spectrum conditions, stratification by sex is essential.

Expression profiling of fibroblasts identifies cell cycle abnormalities in schizophrenia.

Many previous studies have attempted to gain insight into the underlying pathophysiology of schizophrenia by studying postmortem brain tissues of schizophrenia patients. However, such analyses can be confounded by artifactual features of this approach such as lengthy agonal state and postmortem interval times. As several aspects of schizophrenia are also manifested at the peripheral level in proliferating cell types, we have studied the disorder through systematic transcriptomic and proteomic analyses of skin fibroblasts biopsied from living patients. We performed comparative transcriptomic and proteomic profiling to characterize skin fibroblasts from schizophrenia patients compared to healthy controls. Transcriptomic profiling using cDNA array technology showed that pathways associated with cell cycle regulation and RNA processing were altered in the schizophrenia subjects (n = 12) relative to controls (n = 12). LC-MS(E) proteomic profiling led to identification of 16 proteins that showed significant differences in expression between schizophrenia (n = 11) and control (n = 11) subjects. Analysis in silico revealed that these proteins were also associated with proliferation and cell growth pathways. To validate these findings at the protein level, fibroblast protein extracts were analyzed by Western blotting which confirmed the differential expression of three key proteins associated with these pathways. At the functional level, we confirmed the decreased proliferation phenotype by showing that cultured fibroblasts from schizophrenia subjects (n = 5) incorporated less (3)H-thymidine into their nuclei compared to those from controls (n = 6) by day 4 over an 8 day time course study. Similar abnormalities in cell cycle and growth pathways have been reported to occur in the central nervous system in schizophrenia. These studies demonstrate that fibroblasts obtained from living schizophrenia subjects show alterations in cellular proliferation and growth pathways. Future studies aimed at characterizing such pathways in fibroblasts and other proliferating cell types from schizophrenia patients could elucidate the molecular mechanisms associated with the pathophysiology of schizophrenia and provide a useful model to support drug discovery efforts.

Identification of early proteomic markers for hepatic steatosis.

The identification of biomarkers for disease state, drug efficacy, and toxicity is becoming increasingly important for drug discovery and development. We have used two-dimensional differential in-gel electrophoresis and mass spectrometry to identify proteomic markers associated with hepatocellular steatosis in rats after dosing with a compound (CDA) in preclinical development. Rats were dosed daily for up to 5 days with CDA for measurement of blood biochemical parameters, histological, and proteomic analysis. Alterations in plasma glucose and liver transaminases were detected from dosing day 3 onward, and livers showed trace levels of hepatocellular vacuolation from 6 h which increased in extent and severity over the 5 day time course. The number of significantly altered protein spots increased over the 5 day time course, and Ingenuity Pathway Analysis showed that the predominant functions altered by CDA treatment were cell death and cellular assembly and organization. This included alterations in secreted proteins, endoplasmic reticulum and mitochondrial chaperones, antioxidant proteins, and enzymes involved in fatty acid biosynthesis. Comparative in vitro dosing studies showed similar alterations to the proteome, neutral lipid accumulation, and mitochondrial dehydrogenase activity in response to CDA treatment of cultured rat hepatocytes. The finding that several proteins showed significant changes in abundance before the onset of overt toxicity in vivo suggested that these could serve as predictive biomarkers of compounds with a propensity to induce liver steatosis. These markers underwent further direct analysis in the in vitro hepatocyte toxicity model to determine their utility in the development of high throughput assays for drug-induced steatosis.

Proinsulin processing in the diabetic Goto-Kakizaki rat.

The biosynthesis and processing of proinsulin was investigated in the diabetic Goto-Kakizaki (GK) rat. Immunofluorescence microscopy comparing GK and Wistar control rat pancreata revealed marked changes in the distribution of alpha-cells and pronounced beta-cell heterogeneity in the expression patterns of insulin, prohormone convertases PC1, PC2, carboxypeptidase E (CPE) and the PC-binding proteins 7B2 and ProSAAS. Western blot analyses of isolated islets revealed little difference in PC1 and CPE expression but PC2 immunoreactivity was markedly lower in the GK islets. The processing of the PC2-dependent substrate chromogranin A was reduced as evidenced by the appearance of intermediates. No differences were seen in the biosynthesis and post-translational modification of PC1, PC2 or CPE following incubation of islets in 16.7 mM glucose, but incubation in 3.3 mM glucose resulted in decreased PC2 biosynthesis in the GK islets. The rates of biosynthesis, processing and secretion of newly synthesized (pro)insulin were comparable. Circulating insulin immunoreactivity in both Wistar and GK rats was predominantly insulin 1 and 2 in the expected ratios with no (pro)insulin evident. Thus, the marked changes in islet morphology and PC2 expression did not impact the rate or extent of proinsulin processing either in vitro or in vivo in this experimental model.

Identification and characterization of a truncated variant of the 5-hydroxytryptamine(2A) receptor produced by alternative splicing.

We have identified an alternatively spliced 5-hydroxytryptamine 2A receptor (5-HT(2A)-R) transcript by PCR of human brain cDNA using degenerate oligonucleotide primers to transmembrane (TM) domains 3 and 7 of the 5-HT(2)-R subfamily. The variant contains a 118-bp insertion at the exon II/III boundary of the 5-HT(2A)-R, which produces a frame shift in the coding sequence and a premature stop codon. PCR analysis showed that the truncated receptor (5-HT(2A-tr)) and native 5-HT(2A)-R were co-expressed in most brain tissues, with the highest levels being found in hippocampus, corpus collosum, amygdala and caudate nucleus. Western blot analysis of HEK-293 cells transfected transiently with a 5-HT(2A-tr) construct showed that a 30-kDa protein was expressed on cell membranes. Co-transfection studies showed no effect of the 5-HT(2A-tr) variant on 3H-ketanserin binding to the native 5-HT(2A)-R or on functional coupling of the 5-HT(2A)-R to 5-HT-stimulated Ca(2+) mobilization. The functional significance of the 5-HT(2A-tr) variant and other truncated receptors remains to be established.

A PC12 variant lacking regulated secretory organelles: aberrant protein targeting and evidence for a factor inhibiting neuroendocrine gene expression.

A variant of the PC12 pheochromocytoma cell line (termed A35C) has been isolated that lacks regulated secretory organelles and several constituent proteins. Northern and Southern blot analyses suggested a block at the transcriptional level. The proprotein-converting enzyme carboxypeptidase H was synthesised in the A35C cell line but was secreted by the constitutive pathway. Transient transfection of A35C cells with cDNAs encoding the regulated secretory proteins dopamine beta-hydroxylase and synaptotagmin I resulted in distinct patterns of mistargeting of these proteins. It is surprising that hybrid cells created by fusing normal PC12 cells with A35C cells exhibited the variant phenotype, suggesting that A35C cells express an inhibitory factor that represses neuroendocrine-specific gene expression.

Endoplasmic reticulum Ca2+ is important for the proteolytic processing and intracellular transport of proinsulin in the pancreatic beta-cell.

The role of intracellular Ca2+ in the proteolytic processing and intracellular transport of secretory granule proproteins was investigated by pulse-chase radiolabelling of isolated rat islets of Langerhans. The conversion of proinsulin was inhibited by depletion of medium Ca2+ with EGTA and by blocking the transport of Ca2+ into cells with the Ca2+-channel antagonists verapamil, nifedipine and NiCl2. Proinsulin conversion was also reduced by the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin, indicating that the process requires transport of Ca2+ into the endoplasmic reticulum. This was supported by the finding that proinsulin processing was inhibited when Ca2+ was depleted before or during pulse-labelling, but not after transport of the protein to post-endoplasmic-reticulum compartments. Similarly, the inhibition of proinsulin processing was reversed by re-introduction of medium Ca2+ around the time of radiolabelling, but not after 15 min of chase incubation. Ca2+ depletion also decreased proteolytic maturation of the prohormone convertases PC1, PC2 and carboxypeptidase H. Secretion experiments suggested that the rate and extent of proinsulin transport into secretory granules were inhibited marginally by Ca2+ depletion, whereas those of the convertases were markedly impeded. Inhibition of proinsulin conversion by Ca2+ depletion was thus not simply related to the Ca2+-dependencies of mature PC1 and PC2, but also to a requirement for endoplasmic reticulum Ca2+ in proteolytic maturation of the convertases and in their transfer to secretory granules. The results also suggest that the Ca2+ required for prohormone processing in the granules enters the secretory pathway via the endoplasmic reticulum.

The post-translational processing and intracellular sorting of carboxypeptidase H in the islets of Langerhans.

The post-translational processing and intracellular sorting of the proinsulin-converting enzyme carboxypeptidase H (CPH) was studied in isolated rat islets of Langerhans. Pulse-chase-radiolabelling experiments using sequence-specific antisera showed that CPH was synthesized initially as a 57-kDa glycoprotein which was processed to a 54-kDa mature form by proteolytic processing at the N-terminus. Processing of the CPH precursor occurred rapidly (t(1/2) = 30) after an initial delay of 15-30 min and the enzyme was secreted in parallel with the insulin-related peptides in response to glucose-stimulation within 1 h after radiolabelling. This indicated that the proteins were packaged into nascent secretory granules at approximately the same rate following synthesis. Conversion of proinsulin and the 57-kDa form was inhibited markedly by chase incubation of islets at 20 degrees C, indicating that maturation of both proteins occurs in a post-Golgi compartment. Affinity purification of the enzyme from insulinoma subcellular fractions showed that the 57-kDa form was associated with endoplasmic reticulum or Golgi elements, and the 54-kDa form was present in secretory granules. Structural analysis showed that the granule form of the enzyme had an N-terminal amino acid sequence beginning at residue 42 of rat CPH, thereby implicating cleavage of the precursor after the fourth Arg in a site containing five consecutive Arg residues. These findings indicate that post-translational processing of CPH is mediated by an endoprotease which cleaves at sites containing multiple basic amino acid residues upon segregation of the enzyme to the secretory granules.

Differences between the catalytic properties of recombinant human PC2 and endogenous rat PC2.

Human prohormone convertase PC2 was expressed in Xenopus oocytes and its properties were compared with those of the Type-2 endopeptidase of rat insulin secretory granules, previously identified as PC2 [Bennett, Bailyes, Nielson, Guest, Rutherford, Arden and Hutton (1992) J. Biol. Chem. 267, 15229-15236]. Recombinant PC2 had the same substrate specificity as the Type-2 endopeptidase, cleaving at the CA-junction (Lys64, Arg65) of human des-31,32-proinsulin to generate insulin; little activity was found toward human des-64,65-proinsulin or proinsulin itself. Recombinant PC2 was maximally active in 5-7 mM Ca2+ (K0.5 = 1.6 mM) whereas the Type-2 endopeptidase was maximally active in 0.5-1 mM Ca2+ (K0.5 = 40 microM). Both enzymes had a pH optimum of 5.0-5.5 but the Type-2 endopeptidase was active over a wider pH range. Two molecular forms of recombinant PC2 (71 kDa and 68 kDa) were found, both had an intact C-terminus but differed by the presence of the propeptide. The endogenous PC2 comprised several overlapping forms (size range 64-68 kDa), approximately two-thirds of which lacked C-terminal immunoreactivity. Part of the size difference between recombinant and endogenous PC2 was attributable to differences in N-glycosylation. The different post-translational proteolytic modifications of recombinant and endogenous PC2 did not account for the different pH and Ca2+ sensitivities shown by the enzymes. A modulating effect of carbohydrate on enzyme activity could not be excluded.